CN102604847B - Brewing yeast engineering bacteria containing Lg-ATF1 genes - Google Patents
Brewing yeast engineering bacteria containing Lg-ATF1 genes Download PDFInfo
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Abstract
The invention relates to brewing yeast engineering bacteria containing Lg-ATF1 genes; a method for preparing the yeast engineering bacteria provided by The invention comprises the following steps: selecting strong promoters PGK1 so as to over express specific Lg-ATF1 genes coding alcohol acetyl transferase of beer yeast in brewing yeast, thereby obtaining the yeast engineering bacteria SaccharomycescerevisiaeEY-15 containing Lg-ATF1 genes, wherein a preservation number is CGMCC (China General Microbiological Culture Collection Center) No.5635. According to The invention, on a condition that other fermenting performance is not influenced, transformant bacterial strains are compared with parent strains (SaccharomycescerevisiaeCGMCC No.1525); after simulation of semi-solid fermentation, the content of ethyl acetate is increased by 1.6 times; the content of isoamyl acetate is increased to 26.6mg/L; the sifted engineering bacteria has no specific demand for fermentation equipment and conditions; the equipment and conditions of normal white sprits factories can be used; therefore, the engineering bacteria has wide application propose and brings marked economic benefit to industrial production of the white sprits factories.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to the breeding of industrial microorganism, especially a strain contains the saccharomyces cerevisiae engineered yeast of Lg-ATF1 gene.
Background technology
How to improve the content of ester Studies of The Aromatic Substances in wine, be the important topic of China's common white spirit, Yellow Rice Wine Enterprises and the research of related scientific research unit always.The main method that improves at present the common white spirit ester content has following three kinds: the one, and solid-liquid combination method is produced the wine base with liquid phase process, improves quality with vinasse, wine tail or the finished wine of solid state process; The 2nd, the blending method forms to carry out blending with natural perfume modulation or with the pure chemistry medicine by the flavour ingredient of a certain famous brand of wine; The 3rd, full liquid method adds aroma-producing microbe in karusen, the caproic acid bacteria fermented liquid or with caproic acid fermentation liquid through chemistry, after the biological process esterification, then be added in karusen.These methods that improve ester Studies of The Aromatic Substances content in wine are carried out from state of the art mostly, although ester content improves, it is still very large that vinosity and high-grade famous brand of wine differ, and particularly has potential safety hazard adding of pharmaceutical chemicals.The product ester ability that therefore, fundamentally solve yeast saccharomyces cerevisiae still needs to utilize the molecular biology breeding technique to build the Saccharomyces cerevisiae of high ester yield.
Studies show that acetate esters as ethyl acetate (solvent based fragrance) and Isoamyl Acetate FCC (banana flavor), is flavored active ester main in wine.The formation of these ester classes is synthetic in yeast when yeast metabolism, and in fermented liquid, adsorbed by yeast, stays in cell paste by a part by cellular invasion for the ester part of formation.The ester that the content that improves these acetic ester not only can be promoted wine is fragrant, can effectively expand simultaneously, loose nerve the side effect that can reduce drinks causes.
Participating in the synthetic enzyme of acetic ester is mainly alcohol acetyltransferase (AATase), and it can catalytic alcohol and acetyl-CoA formation acetic ester.This enzyme is a kind of sulfydryl enzyme, and three kinds of different types are arranged: AATase I, Lg-AATase I and AATase II, and respectively by ATF1, Lg-ATF1 and ATF2 coding.Yeast saccharomyces cerevisiae only contains an ATF1 gene, and cereuisiae fermentum also contains a homologous gene Lg-ATF1 gene except containing the ATF1 gene.And domesticly also there is no alcohol acetyltransferase and encoding gene thereof to the correlative study of white wine and rice wine flavor and quality impact.
Summary of the invention
The objective of the invention is to solve yeast saccharomyces cerevisiae self and produce the lower problem of ester ability, provide a strain to contain the saccharomyces cerevisiae engineered yeast of Lg-ATF1 gene.
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) engineering bacteria that contains the Lg-ATF1 gene provided by the invention, be specially EY-15, be preserved in Chinese microbial preservation management committee's common micro-organisms center (being called for short CGMCC) on December 22nd, 2011, the address: No. 3, Da Tun road, Chaoyang District, BeiJing, China city first, preserving number is CGMCC No 5636.
Described saccharomyces cerevisiae engineered yeast, in the impregnable situation of other leavening property, after the simulation semi-solid ferment, the transformant bacterial strain is compared with parent strain (Saccharomyces cerevisiae CGMCC No 2.1525), ethyl acetate content improves 1.6 times, and the content of Isoamyl Acetate FCC is brought up to 26.6mg/L.
The construction process of the saccharomyces cerevisiae engineered yeast of the Lg-ATF1 of containing gene of the present invention is:
1) the PGK1 promotor is connected with the pUC19 plasmid with terminator obtains pUC-PGK1;
The homologous fragment IAH that 2) will derive from yeast saccharomyces cerevisiae is connected to the 1st) on the pUC-PGK1 that obtains of step, obtain pUC-PGK1-IAH;
3) coding alcohol acetyltransferase Lg-ATF1 gene in cereuisiae fermentum is inserted into the 2nd) in the pUC-PGK1-IAH that obtains of step between PGK1 promotor and terminator, obtain pUC-PGK1-IAH-Lg-ATF1;
4) the kan gene is connected to the 3rd) go on foot on the pUC-PGK1-IAH-Lg-ATF1 that obtains, obtain plasmid pUC-PGK1-IAH-Lg-ATF1-kan (hereinafter to be referred as pUC-PILgK);
5) with the 4th) the expression plasmid pUC-PILgK that crosses that builds of step cuts with the Bpu1102I enzyme, is inserted into respectively yeast saccharomyces cerevisiae a type and α type monoploid with the Lithium Acetate conversion method, obtains the genes of brewing yeast engineering haploid strains after homologous recombination;
6) the yeast saccharomyces cerevisiae a type after purifying and α type restructuring monoploid are merged, obtain containing the saccharomyces cerevisiae engineered yeast (amphiploid) of Lg-ATF1 gene by resistant panel and living spore experiment screening.
The present invention provides a kind of gene order of identifying the saccharomyces cerevisiae engineered yeast that contains the Lg-ATF1 gene that is specifically designed to simultaneously, this gene order is take Lg-U and Lg-D as primer, take the described saccharomyces cerevisiae engineered yeast strain gene group of Lg-ATF1 gene that contains as template, the amplified fragments order-checking is a specific sequence, as shown in sequence table 1.
Simulation semi-solid ferment: yeast is accessed in 5mL wort nutrient solution 30 ℃ of incubated overnight 12h; Bacterium liquid is all gone in the new sweet wort nutrient solution of 20mL, cultivate 24h for 30 ℃.Get the water that the 100g polished rice is placed in 25～30 ℃, dipping 72h; Take out afterwash, atmospheric cooking 30min.With the rice spreading for cooling, drop in the 500mL triangular flask, add the ripe wheat koji of 10g and 105mL water.At last, access 25mL yeast wort nutrient solution, 28 ℃ fermented 5 days.Fermented liquid is analyzed, comprised that weightlessness, wine degree, residual sugar and GC measure fragrance component content.
GC analyzes: fermented liquid adopts gas chromatography determination after distillation extraction.Internal standard substance is n-amyl acetate.Gas chromatograph is Agilent 7890C; 260 ℃ of chromatographic column HPINNOWAX Polyethylene Glyco: 30m * 320 μ m * 0.5 μ m joins fid detector.Carrier gas is High Purity Nitrogen, flow velocity 2.0mL/min.Initial column temperature is 52 ℃, rises to 70 ℃ with the heat-up rate of 2 ℃/min, then rises to 90 ℃ with the heat-up rate of 4 ℃/min, and the heat-up rate with 10 ℃/min rises to 200 ℃ at last.Detector temperature is 250 ℃, and injector temperature is 230 ℃, and sample size is 1.0 μ L.Shunting mode is shunting, and splitting ratio is 25: 1.
Advantage of the present invention and positively effect:
The present invention selects strong promoter PGK1 that the Lg-ATF1 gene of distinctive coding alcohol acetyltransferase in cereuisiae fermentum is crossed in yeast saccharomyces cerevisiae and expresses; obtain containing the saccharomyces cerevisiae engineered yeast Saccharomyces cerevisiae EY-15 of Lg-ATF1 gene, preserving number is CGMCC No 5636.
The saccharomyces cerevisiae engineered yeast Saccjaromyces cerevisiae EY-15 (preserving number CGMCC No 5636) that contains the Lg-ATF1 gene that the present invention obtains compares with initial S. cervisiae (recipient bacterium Saccharomyces cerevisiae CGMCCNo 2.1525): after the simulation semi-solid ferment, ethyl acetate content improves 1.6 times, the content of Isoamyl Acetate FCC is brought up to 26.6mg/L, for wine industry production provides strain excellent.
Description of drawings
The checking electrophorogram of Fig. 1 .pUC-PILgK plasmid.
Fig. 2. the haploid checking of positive recombinant Saccharomyces cerevisiae, wherein (a) is the positive restructuring of a type monoploid the result; (b) be the positive restructuring of α type monoploid the result.
Fig. 3. contain the structure route map of the saccharomyces cerevisiae engineered yeast of Lg-ATF1 gene.
The saccharomyces cerevisiae engineered yeast (Saccharomyces cerevisiae) that a strain of the present invention contains the Lg-ATF1 gene is specially EY-15, be preserved in Chinese microbial preservation management committee's common micro-organisms center (being called for short CGMCC) on December 22nd, 2011, the address is: No. 3, Da Tun road, Chaoyang District, BeiJing, China city first, preserving number is CGMCC No 5636.
Embodiment
Yeast saccharomyces cerevisiae amphiploid thalline used in the present invention is the yeast saccharomyces cerevisiae amphiploid bacterial strain that can adopt any source.
Method in following embodiment if no special instructions, is ordinary method.
Embodiment 1: the structure that contains the saccharomyces cerevisiae engineered yeast of Lg-ATF1 gene
(1) structure of engineering strain
1) the PGK1 promotor is connected with the pUC19 plasmid with terminator obtains pUC-PGK1;
The homologous fragment IAH that 2) will derive from yeast saccharomyces cerevisiae is connected to the 1st) on the pUC-PGK1 that obtains of step, obtain pUC-PGK1-IAH;
3) coding alcohol acetyltransferase Lg-ATF1 gene in cereuisiae fermentum is inserted into the 2nd) in the pUC-PGK1-IAH that obtains of step between PGK1 promotor and terminator, obtain pUC-PGK1-IAH-Lg-ATF1;
4) the kan gene is connected to the 3rd) go on foot on the pUC-PGK1-IAH-Lg-ATF1 that obtains, obtain plasmid pUC-PGK1-IAH-Lg-ATF1-kan (hereinafter to be referred as pUC-PILgK);
Fig. 1 is the checking electrophorogram of pUC-PILgK plasmid: wherein swimming lane 1 is 5000 bp DNA Ladder Marker; Swimming lane 2 is recipient bacterium Genomic PCR amplification homology segment IAH; Swimming lane 3 is pUC-PILgK plasmid PCR amplification homology segment IAH; Swimming lane 4 is beer yeast gene group pcr amplification Lg-ATF1; Swimming lane 5 is pUC-PILgK plasmid PCR amplification Lg-ATF1; Swimming lane 6 is pUG6 plasmid PCR amplification Kan; Swimming lane 7 is pUC-PILgK plasmid PCR amplification Kan; Swimming lane 8 is the pUC-PILgK plasmid, PGK1 upstream primer+downstream primer pcr amplification result; Swimming lane 9 is 1Kb DNALadder Marker; Swimming lane 10 is the linear result of carrier pUC19 single endonuclease digestion; Swimming lane 11 is the linear result of pUC-PILgK plasmid single endonuclease digestion.
5) Bpu 1102I (Blp I) digested plasmid pUC-PILgK; Be inserted into respectively yeast saccharomyces cerevisiae (Saccharomyces cerevisiae CGMCC No 2.1525) a type and α type monoploid with the Lithium Acetate conversion method, obtain the genes of brewing yeast engineering haploid strains after homologous recombination.In Fig. 2, (a) is the positive restructuring of a type monoploid PCR the result; (b) be the positive restructuring of α type monoploid PCR the result.Wherein swimming lane 1 is 5000bp DNALadder Marker, and swimming lane 2 is recipient bacterium a/ α type monoploid upstream PCR negative control; Swimming lane 3 is a/ α type restructuring monoploid upstream PCR product; Swimming lane 4 is recipient bacterium a/ α type monoploid downstream PCR negative control; Swimming lane 5 is a/ α type restructuring monoploid downstream PCR product.
6) the yeast saccharomyces cerevisiae a type after purifying and α type restructuring monoploid are merged, obtain containing the saccharomyces cerevisiae engineered yeast EY-15 (amphiploid) of Lg-ATF1 gene by resistant panel and living spore experiment screening.Fig. 3 is the building process that contains the saccharomyces cerevisiae engineered yeast of Lg-ATF1 gene.
(2) specific sequence of engineering strain
Contain one section specific sequence in the engineering strain EY-15 karyomit(e) of above acquisition, carry out identification of strains after can checking order by pcr amplification.
The primer sequence of specific fragment amplification is:
Lg-U：5’-GGAGGAGACCGAACACAAGTATC-3’
Lg-D：5’-TGGTTTGGAGGAGAAGATAACGACG-3’
The gene order of this specific fragment is seen sequence table 1.
Embodiment 2: contain the research of saccharomyces cerevisiae engineered yeast and the starting strain leavening property of Lg-ATF1 gene
Engineering bacteria and recipient bacterium are accessed respectively in 5mL wort nutrient solution 30 ℃ of incubated overnight 12h; Bacterium liquid is all gone in the new sweet wort nutrient solution of 20mL, cultivate 24h for 30 ℃.Get the water that the 100g polished rice is placed in 25～30 ℃, dipping 72h; Take out afterwash, atmospheric cooking 30min.With the rice spreading for cooling, drop in the 500mL triangular flask, add the ripe wheat koji of 10g and 105mL water.At last, access 25mL yeast wort nutrient solution, 28 ℃ fermented 5 days.Vibrate and weigh every 12h between yeast phase, record is weightless; After fermentation ends, stop cultivating and weighing; Measure remaining sugar concentration, alcohol volume fraction and the key odorant component content of fermented liquid, characterize its over-all properties with fermentation capacity, remaining sugar concentration and product growing amount, the results are shown in Table 1.
The leavening property of table 1 yeast saccharomyces cerevisiae recipient bacterium and engineering bacteria
Annotate: shown in data be the mean value of three parallel test results.
Embodiment 3: contain the engineering monoploid of Lg-ATF1 gene and the research of starting strain monoploid leavening property
Engineering monoploid (a/ α type) and recipient bacterium monoploid (a/ α type) are accessed respectively in 5mL wort nutrient solution 30 ℃ of incubated overnight 12h; Bacterium liquid is all gone in the new sweet wort nutrient solution of 20mL, cultivate 24h for 30 ℃.Get the water that the 100g polished rice is placed in 25～30 ℃, dipping 72h; Take out afterwash, atmospheric cooking 30min.With the rice spreading for cooling, drop in the 500mL triangular flask, add the ripe wheat koji of 10g and 105mL water.At last, access 25mL yeast wort nutrient solution, 28 ℃ fermented 5 days.Vibrate and weigh every 12h between yeast phase, record is weightless; After fermentation ends, stop cultivating and weighing; Measure remaining sugar concentration, alcohol volume fraction and the key odorant component content of fermented liquid, characterize its over-all properties with fermentation capacity, remaining sugar concentration and product growing amount, the results are shown in Table 2.
Table 2 yeast saccharomyces cerevisiae recipient bacterium monoploid and the haploid leavening property of engineering
Annotate: shown in data be the mean value of three parallel test results.
Claims (2)
1. a strain contains yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) engineering bacteria of Lg-ATF1 gene, is specially EY-15, and preserving number is CGMCC No5636.
2. saccharomyces cerevisiae engineered yeast according to claim 1, after it is characterized in that simulating semi-solid ferment, the transformant bacterial strain is compared with parent strain yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No2.1525, ethyl acetate content improves 1.6 times, and the content of Isoamyl Acetate FCC is brought up to 26.6mg/L.
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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 |
| CN108485996B (en) * | 2018-05-18 | 2020-06-30 | 天津科技大学 | Novel ethyl acetate-producing saccharomyces cerevisiae strain and construction method thereof |
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| EP0977868A1 (en) * | 1998-02-05 | 2000-02-09 | Hoechst Marion Roussel | Yeast strains with interrupted atf2 gene and uses |
| CN102186988A (en) * | 2008-07-26 | 2011-09-14 | 诺丁汉大学 | Novel methods of differentiating yeast strains and/or determining genetic stability of yeast strains, and uses thereof |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0977868A1 (en) * | 1998-02-05 | 2000-02-09 | Hoechst Marion Roussel | Yeast strains with interrupted atf2 gene and uses |
| CN102186988A (en) * | 2008-07-26 | 2011-09-14 | 诺丁汉大学 | Novel methods of differentiating yeast strains and/or determining genetic stability of yeast strains, and uses thereof |
| 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 |
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