CN109234303A - The construction method of phenylpyruvate decarboxylase Aro10 is expressed in a kind of brewer's yeast - Google Patents

The construction method of phenylpyruvate decarboxylase Aro10 is expressed in a kind of brewer's yeast Download PDF

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CN109234303A
CN109234303A CN201811186280.4A CN201811186280A CN109234303A CN 109234303 A CN109234303 A CN 109234303A CN 201811186280 A CN201811186280 A CN 201811186280A CN 109234303 A CN109234303 A CN 109234303A
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aro10
yeast
brewer
gene
pypge
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钟成
丁霄彤
张凯月
刘伶普
贾士儒
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Tianjin University of Science and Technology
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Tianjin University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01043—Phenylpyruvate decarboxylase (4.1.1.43)

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  • Genetics & Genomics (AREA)
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  • Wood Science & Technology (AREA)
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  • Physics & Mathematics (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Distillation Of Fermentation Liquor, Processing Of Alcohols, Vinegar And Beer (AREA)

Abstract

It is an object of the invention to construct a kind of method that phenylpyruvate decarboxylase Aro10 is expressed in brewer's yeast, specific step is as follows: (1) acquisition of beer yeast gene group: 3rd area brewer's yeast TT21 is cultivated, until monoclonal out, then it puts down to be coated on plate and obtains thallus, handled with SDS and obtain beer yeast gene group；(2) clone of target gene: utilizing reasonable primer, condition, and PCR amplification goes out the gene of Aro10 (S.c), Aro10 (S.b)；(3) construction of expression vector: using pYPGE15 plasmid as carrier, Aro10 (S.c), Aro10 (S.b) gene are inserted into the downstream promoter PGK respectively, construct pYPGE-ARO10 (S.c), pYPGE-ARO10 (S.b) expression vector；(4) it obtains recombinant bacterial strain: pYPGE-ARO10 (S.c), pYPGE-ARO10 (S.b) expression vector being transferred in brewer's yeast TT21 by lithium acetate chemical transformation, obtain the recombination yeast of Aro10；(5) beer fermentation of recombinant bacterial strain；(6) benzyl carbinol content detection: detect using high performance liquid chromatography the variation of fragrant alcohol content.

Description

The construction method of phenylpyruvate decarboxylase Aro10 is expressed in a kind of brewer's yeast
Technical field
The present invention relates to a kind of recombination brewer's yeast systems for expressing phenylpyruvate decarboxylase Aro10 to improve aromatic alcohol flavor Building, belong to gene engineering technology field.
Background technique
Brewer's yeast can use various amino acid as nitrogen source, including three kinds of ArAA l-tyrosine, L-phenylalanine and L-Trp.Its main metabolites is tyrosol, benzyl carbinol and tryptophol, they are referred to as fusel oil.From ammonia The fusel oil that base acid is formed is changed by three kinds of enzymatics of Ehrlich pathway.In the example of phenylalanine, amino acid is de- Amino becomes phenylpyruvic acid, and then decarboxylation becomes phenylacetaldehyde and forms benzyl carbinol.
In the generation of beer, extremely important effect is played in the forming process of flavor substance.Fusel oil is in its production The product of formation, higher alcohol (fusel oil) are the most volatile flavor substances of content.Higher alcohol includes propyl alcohol, 2- methylpropanol (isobutanol), optically active amyl alcohol, 3- methyl butanol (isoamyl alcohol) and benzyl carbinol, benzyl carbinol are the important composition portions of fusel oil Point, and determine a key factor of fermented food quality.
Benzyl carbinol (Phenethyl Alcohol) be colourless transparent liquid, have Rose type fragrance, the simple and elegant exquisiteness of fragrance, The soft sweet tea of fragrance and.Its flavor characteristic recalling rose honey, has broad application prospects in food, fermentation, brewing industry. Benzyl carbinol is the important aroma compound of the alcoholic beverage such as pure mellow wine and grape wine.For example, containing 20-70mg benzene in every 1L pure mellow wine Ethyl alcohol.It is higher than 100mg/L in yellow rice wine.Therefore, in order to increase the rich in taste degree of beer, the content of aromatic alcohol can suitably be changed.
The forming feature of aromatic alcohol has two:
1, amino acid degradation metabolic pathway: from amino acid metabolism route it is found that in general, when certain monoamino-acid in wheat juice Content exceed when needing of yeast, then extra amino acid is formed higher alcohol.
2, fermentable sugar metabolic pathway of synthesizing: from glycometabolism route it is found that the final stage shape of biosynthesis amino acid At 2-ketoacid, effect of the 2-ketoacid through decarboxylase and alcohol dehydrogenase generates corresponding aromatic alcohol.
In the metabolic regulation of aromatic alcohol the first step react transaminase and thallus physiological growth it is closely related, by with Transamination between α-ketoglutaric acid is closely connected with TCA circulation.But most researchers are concerned with second at present Phenylpyruvate decarboxylase in step reaction, it is the key enzyme in Emhorn approach.Under conditions of different nitrogen sources, phenylpyruvic acid decarboxylation Enzymatic activity is mainly gene-determined by Aro10.
Summary of the invention
It is an object of the invention to construct a kind of recombination brewer's yeast for expressing phenylpyruvate decarboxylase Gene A ro10.
It is provided by the invention expression Aro10 brewer's yeast system specific steps such as:
(1) acquisition of beer yeast gene group: 3rd area brewer's yeast TT21 is cultivated, until monoclonal out, then puts down and apply A large amount of thallus are obtained on plate, obtain beer yeast gene group with SDS processing thallus；
(2) clone of target gene: utilizing reasonable homologous primer, appropraite condition, and PCR amplification goes out Aro10 (S.c), Aro10 (S.b) gene；
(3) construction of expression vector: using pYPGE15 plasmid as carrier, Aro10 (S.c), Aro10 (S.b) gene are inserted respectively Enter the downstream promoter PGK, 5 ' end restriction enzyme sites are BmaH I, and 3 ' end restriction enzyme sites are Sma I, construct pYPGE-ARO10 (S.c), pYPGE-ARO10 (S.b) expression vector；
(4) it obtains recombinant bacterial strain: pYPGE-ARO10 (S.c), pYPGE-ARO10 (S.b) expression vector is passed through into lithium acetate Chemical transformation is transferred in brewer's yeast TT21, obtains the recombination yeast of Aro10；
(5) beer fermentation of recombinant bacterial strain；
(6) benzyl carbinol content detection: fragrant alcohol content is measured using high performance liquid chromatography, detector is diode Array detector detects the variation of fragrant alcohol content.
Positive effect of the invention
1. the present invention, using the method carrier construction for using plasmid expression gene, the method for being relatively integrated into genome is more simple Just quickly, building week is greatly shortened.
2. the present invention expresses the Aro10 gene in brewer's yeast, the primer sequence of target gene is optimized, rationally Aro10 gene is added, brewer's yeast is made to supplement other genes without external source.
5. the present invention obtains extraction beer yeast gene group, using solid medium mass propgation thallus.
6. the present invention realizes overexpression of the phenylpyruvate decarboxylase Gene A ro10 in yeast, recombination beer is improved Saccharomycete promotes the industrial production potentiality of recombination saccharomyces cerevisiae, flavor is promoted in beer fermentation in aromatic alcohol generation efficiency
Detailed description of the invention
Fig. 1 is the pcr amplification figure of Aro10 (S.c), Aro10 (S.b)；
Fig. 2 is the E. coli clones proof diagram of pYPGE-ARO10 (S.c), pYPGE-ARO10 (S.b)；
Fig. 3 is pYPGE-ARO10 (S.c), pYPGE-ARO10 (S.b) expression plasmid map；
Fig. 4 is pYPGE-ARO10 (S.c), pYPGE-ARO10 (S.b) expression plasmid digestion verification figure；
Fig. 5 is recombination brewer's yeast TT21/pYPGE-ARO10-B, TT21/pYPGE-ARO10-C proof diagram；
Fig. 6 is benzyl carbinol containing spirogram.
Specific embodiment
The clone of 1 target gene of case study on implementation
1. by the yeast bacteria suspension of glycerol tube is stored in, is taken out from -80 DEG C of refrigerators and be placed in mixture of ice and water (or 4 DEG C of ice Case) in thaw, until yeast bacteria suspension melts completely.Sterilised yeast suspension is uniformly mixed with pipettor, 100 μ L is drawn and is coated on YPD On plate, after being sealed plate with sealed membrane, it is inverted in 25 DEG C of incubators and cultivates.Culture about 2-3 days, until yeast single colonie is long To 2-3mm, it is placed in spare in 4 DEG C of refrigerators.
2. fresh yeast single colonie is closely lined on YPD plate with oese picking, stationary culture, until thallus is long Out.Hypothallus is scraped from plate, and the Ep pipe of the SDS solution containing 30 μ L is added, is mixed, is put to 95 DEG C of water with turbine mixer It is placed on ice after five minutes in bath 5 minutes, centrifugation draws 20 μ L of supernatant into new Ep pipe afterwards in triplicate, and -20 DEG C of placement is standby With.
3. going out gene sequence corresponding to the Aro10 of TT21 by homologous primer PCR amplification using Yeast genome as template Column, amplification PCR figure are shown in Fig. 1.
The building of 2 pYPGE-Aro10 expression vector of case study on implementation
1. first Buffer, restriction enzyme are placed in ice, sufficiently melt to it and after mixing, prepares double digestion Reaction system is uniformly mixed after preparation, is placed in 30 DEG C of water-baths, digestion 30min.After digestion, agarose is used Gel-purified QIAquick Gel Extraction Kit recycles digestion products, does not cut plasmid to remove in digestion products, in order to avoid influence subsequent matter The connection of grain and target gene.
2. 3 ' end Sma I are restriction enzyme site, insertion pYPGE15 plasmid vector starting using 5 ' end BamH I as restriction enzyme site The sub- downstream PGK, construction of expression vector pYPGE-Aro10 expression vector map are shown in Fig. 3.Fig. 2 is shown in E. coli clones PCR verifying. And expression vector is verified by single endonuclease digestion, double digestion, to determine that expression vector pYPGE-Aro10 constructs successfully digestion Proof diagram is shown in Fig. 4.
3 expression vector pYPGE-Aro10 chemical transformation of case study on implementation is transferred in brewer's yeast
1. expression vector pYPGE-Aro10 is transferred in brewer's yeast TT21 by lithium acetate chemical transformation, apply Plate obtains transformant.
2. slightly mentioning the genome for the recombination brewer's yeast transformant that 1 obtains using SDS pyrolysis method, tested by PCR method Its correctness is demonstrate,proved, Genomic PCR nucleic acid figure is shown in Fig. 5.
4 high performance liquid chromatography of case study on implementation verifies fragrant alcohol content in fermentation liquid
1. aseptically, taking in brewer's yeast single colonie access 5mLYPD fluid nutrient medium, 25 DEG C of stationary cultures 48h；Then it takes 100 μ L switching in 10mLYPD fluid nutrient medium, it is spare that 10 DEG C of incubators is put into after 25 DEG C of stationary culture 48h.
2. weighing is gone to take thallus after thalline were collected by centrifugation, the inoculum concentration of 1.1g yeast is inoculated into wheat juice in every 220mL wheat juice In, 12 DEG C of main fermentation temperature, every pol and ethanol content are measured by sampling for 24 hours, until the near 5.0 ° of P of pol, main ferment terminate, take Wine sample is analyzed.
3. the wine sample membrane filtration in 0.22 μm of aperture is carried out high performance liquid chromatography, diode battle array after collecting filtrate Column detector detects fragrant alcohol content, fragrant alcohol content such as Fig. 6.

Claims (5)

1. expressing the construction method of phenylpyruvate decarboxylase Aro10 in a kind of brewer's yeast, comprising the following steps:
(1) acquisition of beer yeast gene group: 3rd area brewer's yeast TT21 is cultivated, until monoclonal out, then put down be coated in it is flat A large amount of thallus are obtained on plate, obtain beer yeast gene group with SDS processing thallus；
(2) clone of target gene: utilizing reasonable homologous primer, appropraite condition, and PCR amplification goes out Aro10 (S.c), Aro10 (S.b) gene；
(3) construction of expression vector: using pYPGE15 plasmid as carrier, Aro10 (S.c), Aro10 (S.b) gene are inserted into open respectively The downstream mover PGK, 5 ' end restriction enzyme sites are BmaH I, and 3 ' hold restriction enzyme sites for Sma I, building pYPGE-ARO10 (S.c), PYPGE-ARO10 (S.b) expression vector；
(4) it obtains recombinant bacterial strain: pYPGE-ARO10 (S.c), pYPGE-ARO10 (S.b) expression vector is passed through into lithium acetate chemistry Conversion method is transferred in brewer's yeast TT21, obtains the recombination yeast of Aro10；
(5) beer fermentation of recombinant bacterial strain；
(6) benzyl carbinol content detection: fragrant alcohol content is measured using high performance liquid chromatography, detector is diode array Detector detects the variation of fragrant alcohol content.
2. expressing the construction method of Aro10 in a kind of brewer's yeast according to claim 1, which is characterized in that use matter The method carrier construction of grain expressing gene, the method for being relatively integrated into genome is more easy to be quick, greatly shortens construction schedule.
3. expressing the construction method of Aro10 gene in a kind of brewer's yeast according to claim 1, which is characterized in that will Aro10 gene in brewer's yeast is expressed, and the primer sequence of target gene is optimized, and Aro10 gene is rationally added, makes beer Brewer yeast supplements other genes without external source.
4. expressing the construction method of Aro10 in a kind of brewer's yeast according to claim 1, which is characterized in that mentioned When taking beer yeast gene group, using solid medium mass propgation thallus.
5. expressing the construction method of Aro10 in a kind of brewer's yeast according to claim 1, which is characterized in that realize Overexpression of the phenylpyruvate decarboxylase Gene A ro10 in yeast, improves recombination saccharomyces cerevisiae in aromatic alcohol generation efficiency, The industrial production potentiality for promoting recombination saccharomyces cerevisiae, promote flavor in beer fermentation.
CN201811186280.4A 2018-10-12 2018-10-12 The construction method of phenylpyruvate decarboxylase Aro10 is expressed in a kind of brewer's yeast Pending CN109234303A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111690638A (en) * 2020-06-12 2020-09-22 天津科技大学 Construction method for overexpression of aromatic amino acid transaminase II Aro9 in beer yeast
CN112646831A (en) * 2019-10-10 2021-04-13 天津科技大学 Shuttle plasmid, construction method and application thereof in synechocystis transformation exogenous gene

Citations (2)

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CN103409332A (en) * 2012-12-14 2013-11-27 北京工商大学 Construction of saccharomyces cerevisiae engineering bacteria generating beta-phenethyl alcohol, strain and application thereof
WO2018067068A1 (en) * 2016-10-07 2018-04-12 National University Of Singapore Method of engineering microorganisms for the overproduction of short branched-chain fatty acids

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN103409332A (en) * 2012-12-14 2013-11-27 北京工商大学 Construction of saccharomyces cerevisiae engineering bacteria generating beta-phenethyl alcohol, strain and application thereof
WO2018067068A1 (en) * 2016-10-07 2018-04-12 National University Of Singapore Method of engineering microorganisms for the overproduction of short branched-chain fatty acids

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Title
YIN,SHENG等: "Improving 2-Phenylethanol Production via Ehrlich Pathway Using Genetic Engineered Saccharomyces cerevisiae Strains", 《CURRENT MICROBIOLOGY》 *
梁婧如等: "脱羧酶基因ARO10的过量表达及其对Saccharomy cescerevisiae的β-苯乙醇合成代谢影响", 《食品工业科技》 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112646831A (en) * 2019-10-10 2021-04-13 天津科技大学 Shuttle plasmid, construction method and application thereof in synechocystis transformation exogenous gene
CN111690638A (en) * 2020-06-12 2020-09-22 天津科技大学 Construction method for overexpression of aromatic amino acid transaminase II Aro9 in beer yeast

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Application publication date: 20190118