CN106119141A - One strain by knocking out CAR1 process LAN DUR1,2 low yield urethanes Wine brewing yeast strain and construction methods thereof - Google Patents

One strain by knocking out CAR1 process LAN DUR1,2 low yield urethanes Wine brewing yeast strain and construction methods thereof Download PDF

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CN106119141A
CN106119141A CN201610020591.8A CN201610020591A CN106119141A CN 106119141 A CN106119141 A CN 106119141A CN 201610020591 A CN201610020591 A CN 201610020591A CN 106119141 A CN106119141 A CN 106119141A
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dur1
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肖冬光
李园子
杜丽平
郭学武
陈叶福
张翠英
董健
马立娟
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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
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/03—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amidines (3.5.3)
    • C12Y305/03001—Arginase (3.5.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G2200/00—Special features
    • C12G2200/11—Use of genetically modified microorganisms in the preparation of wine

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Abstract

The invention discloses strain low yield urethanes saccharomyces cerevisiae and a construction method thereof, belong to technical field of bioengineering.The present invention by parent's Wine brewing yeast strain, knocks out the arginase gene CAR1 of a copy, selects strong promoter PGK1 process LAN urea groups amidase gene DUR1 simultaneously, and 2, obtain low yield urethanes Wine brewing yeast strain.In Wine-making Experiment, compared with parent strain, other fermenting properties of constructed Wine brewing yeast strain and flavor substance are unaffected, and the content of carbamide and EC significantly reduces, and wherein the content of carbamide reduces 40.38%, and the content of EC reduces 34.45%.Meet the wine association area high requirement to yeast, have wide practical use.

Description

One strain by knocking out CAR1 process LAN DUR1,2 low yield urethanes saccharomyces cerevisiaes Bacterial strain and construction method thereof
Technical field:
The invention belongs to technical field of bioengineering, relate to the breeding of industrial microorganism, be specifically related to a strain low yield ammonia The Wine brewing yeast strain of base Ethyl formate and construction method thereof.
Background technology:
Urethanes (Ethyl Carbamate, EC) is a kind of potential carcinogen, may result in pulmonary carcinoma, lymphatic cancer, liver The disease such as cancer and skin carcinoma, within 2007, international cancer research institution reclassifies EC, (maybe may be made us class by 2B group and suffer from cancer Material) change 2A group (material that class suffers from cancer may be made us) into.EC be widely present in fermented food (as bread, clabber, cheese and Soy sauce etc.) and alcoholic beverage (such as Chinese rice wine, sake and wine) in.Wine be the most ancient beverage it One, there is local flavor and the alimentary health-care function of uniqueness.After testing, in wine, EC content is higher.European and American developed countries are the most right EC limit standard formulated by the wine of market sale.Therefore, reduce the EC content in wine, to promoting that Grape Wine Industry is good for Kang Fazhan and guarantee human body health have profound significance.
How to reduce the content of EC in wine, always be international Wine Enterprises and the weight of related scientific research unit research Want problem.EC in wine is mainly formed by carbamide and ethanol spontaneous reaction, and the main producers material of EC is carbamide.Cause This, in the strategy reducing EC content, mainly achieved the goal by the precursor substance carbamide of reduction EC.Reduce at present In wine, the main method of EC content has the following two kinds: one is by adding acid urease degradation of urine in vinous fermentation liquid Element, thus reduce the generation of EC.But owing to the production process of acid urease is loaded down with trivial details, cost is high, and the use of acid urease can significantly Increase enterprise's production cost, and the interpolation of external enzyme there may be the risk of secondary pollution.Two, it is to examine in terms of wine-making technology Consider, suitably change some process conditions and then reduce the generation of EC.But which may cause changing of flavor substance in wine Become, the decline of wine quality.Therefore, the product carbamide ability that will fundamentally reduce saccharomyces cerevisiae needs for utilizing molecule raw Thing breeding technique builds the Saccharomyces cerevisiae of high-yield urea.
Research shows, the carbamide in wine is mainly divided under the effect of the arginase of CAR1 gene code by arginine Solve and produce.When yeast needs nitrogen source, carbamide is decomposed into NH by DUR1, the urea groups amidase of 2 gene codes3And CO2, thus Synthesize various nitrogen-containing compound for self-demand.When intracellular urea content is too high, can be turned by the carbamide of DUR4 gene code Fortune protein D ur4p transports out cell.When yeast needs nitrogen source again, the carbamide in fermentation liquid also can be by DUR3 base on cell membrane Carry because of Urea transport protein Dur3p of coding and proceed to intracellular, heavily absorbed by yeast cells.Therefore, arginase is knocked out Gene C AR1 blocks the source of carbamide, and process LAN decomposing urea is NH3And CO2Urea groups amidase gene DUR1,2 promote carbamide Decomposition be to reduce the effective measures of urea content in wine.Therefore, the present invention is by knocking out the arginase of a copy Gene C AR1, simultaneously process LAN urea groups amidase gene DUR1,2, selection-breeding low yield EC Saccharomyces cerevisiae, meet wine brewing ferment Female application association area high requirement to yeast, to promoting Grape Wine Industry to develop in a healthy way and ensureing that human body health has Profound significance.
Summary of the invention:
One of present invention solves the technical problem that and to be to provide a strain low yield urethanes saccharomyces cerevisiae.
Described low yield urethanes saccharomyces cerevisiae is the arginase knocking out a copy in the yeast strain that sets out Gene C AR1, simultaneously process LAN urea groups amidase gene DUR1,2 gained.
Its Gene ID of described CAR1 gene is: 855993, and nucleotide sequence is as shown in SEQ NO:1 in table；Described DUR1, Its Gene ID of 2 genes is: 852507, and nucleotide sequence is as shown in SEQ NO:2 in table；Its Gene ID of described promoter PGK1 For: 850370, nucleotide sequence is as shown in SEQ ID NO:3 in table.
The described yeast strain that sets out is saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3373, protects Being stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the public is available commercially.
Another technical problem that the present invention solves is to provide the structure of a kind of low yield urethanes Wine brewing yeast strain Construction method, comprises the steps:
(1) recombiant plasmid Yep-APD1,2The structure of KB
1. with plasmid pPGK1 as template, PCR expands strong promoter PGK1 gene, with restricted enzyme BamHI and SalI Vector plasmid YEP352 and PGK1 genetic fragment are carried out enzyme action, then with Solution I ligase, both is connected structure plasmid Yep-P；
2. with the yeast strain genome that sets out as template, PCR expands DUR1,2 genes, confronts with restricted enzyme XhoI Grain Yep-P carries out enzyme action, then with Infusion ligase, DUR1, the plasmid Yep-P after 2 genetic fragments and enzyme action is connected structure Plasmid Yep-PD1,2；
3. with plasmid pUG6 as template, PCR expands KanMX gene, with restricted enzyme BamHI to plasmid Yep-PD1,2 Carry out enzyme action, then with Infusion ligase by the plasmid Yep-PD after KanMX genetic fragment and enzyme action1,2Connect and build plasmid Yep-PD1,2K；
4. with the yeast strain genome that sets out as template, PCR expands DUR1, and the upstream homology arm CUA of 2 genes, with restriction Restriction endonuclease SmaI is to plasmid Yep-PD for property1,2K carries out enzyme action, then with Infusion ligase by DUR1, the upstream homology of 2 genes Plasmid Yep-PD after arm CUA genetic fragment and enzyme action1,2K connects structure plasmid Yep-APD1,2K；
5. with the yeast strain genome that sets out as template, PCR expands DUR1, and the downstream homology arm CUB of 2 genes, with restriction Restriction endonuclease SphI is to plasmid Yep-APD for property1,2K carries out enzyme action, then with Infusion ligase by DUR1, the downstream homology of 2 genes Plasmid Yep-APD after arm CUB genetic fragment and enzyme action1,2K connects structure plasmid Yep-APD1,2KB。
(2) DUR1, the process LAN of 2 genes while of knocking out of CAR1 gene
1. with plasmid Yep-APD1,2KB is template, and PCR expands " CUA-KanMX-PGK1p-DUR1,2-PGK1t-CUB " weight Pack section；
2. being imported by the PCR recombinant fragment of step (2)-1. obtain by lithium acetate transformation method sets out in yeast strain, it is thus achieved that Knock out the CAR1 gene of a copy, process LAN DUR1 simultaneously, the recombinant bacterial strain 1 of 2 genes；
3. the KanMX gene in bacterial strain is obtained with pGAP plasmid removing step (2)-2., it is thus achieved that without resistant gene, knock out The CAR1 gene of one copy, process LAN DUR1 simultaneously, the recombinant bacterial strain 2 of 2 genes, passes on the restructuring obtained without pGAP plasmid Bacterial strain 2.
Described recombinant bacterial strain can be built by said method, involved concrete operation method a lot of document reports, as Joseph Sambrook etc., " Molecular Cloning: A Laboratory guide " second edition, Science Press, 1995.
Saccharomyces cerevisiae recombinant bacterial strain 2 of the present invention, in the case of other fermenting property is unaffected, wine is sent out In ferment test, the content of carbamide reduces 40.38%, and the content of EC reduces 34.45%.
Beneficial effect:
1, the invention provides a kind of low yield urethanes Wine brewing yeast strain, reduce containing of EC in wine Amount, overcomes the impact big on human carcinogen's risk that common saccharomyces cerevisiae causes owing to producing EC content height.
2, the low yield EC saccharomyces cerevisiae that the present invention provides is on the premise of keeping good fermenting property, has knocked out a copy Arginase gene CAR1, urea groups amidase gene DUR1 simultaneously, the expression of 2 significantly improves, and has reached the mesh of low yield EC , establish theoretical basis for brewing the wine being more beneficial for health, there is wide market prospect, simultaneously for grinding Study carefully related gene the impact of carbamide in wine and EC content is laid a good foundation.
3, the yeast strain that selection-breeding of the present invention obtains eliminates KanMX resistant gene, no completely by Cre/LoxP system Containing external source resistant gene, can safety for commercial production, have wide practical use.
Accompanying drawing illustrates:
Fig. 1 is Yep-APD1,2KB plasmid construction process；
Fig. 2 is plasmid Yep-APD1,2The PCR checking of KB:
Wherein: in (a), M is marker；1 is with Yep-APD1,2KB is template, and PGK1-U/PGK1-D is primer PCR amplification PGK1p-DUR1,2-PGK1tFragment；
B in (), M is marker；1 is with Yep-APD1,2KB is template, and CU-U/CU-D is that primer PCR expands CUA- KanMX-PGK1p-DUR1,2-PGK1t-CUB fragment；
Fig. 3 is the DUR1 while of knocking out of CAR1 gene, the checking of the process LAN recombinant bacterial strain of 2 genes:
A in (), M is marker；1 is that U-S/KU-S is primer with the genome of recombinant bacterial strain 1 as template, and PCR amplification is tested Card fragment；2 is that U-S/KU-S is primer with the genome of starting strain as template, PCR amplification checking fragment；3 is with recombinant bacterium The genome of strain 1 is template, and PU-X/U-X is primer, PCR amplification checking fragment；4 is with the genome of starting strain as template, PU-X/U-X is primer, PCR amplification checking fragment；
B in (), M is marker；1 is that 2 is to be with the genome of recombinant bacterial strain 1 with the genome of recombinant bacterial strain 2 as template Template, with Kan-U/Kan-D as primer, PCR amplification checking fragment；
C in (), M is marker；1 for the genome of the recombinant bacterial strain 2 after passing on as template, and 2 is with the restructuring before passing on The genome of bacterial strain 2 is template, with Zeocin-U/Zeocin-D as primer, and PCR amplification checking fragment；
Fig. 4 is 9-hydroxyl ton and carbamide derivatization reaction schematic diagram in acid condition.
Detailed description of the invention:
A kind of low yield EC saccharomyces cerevisiae and selection thereof below by the specific embodiment narration present invention.Following Technological means used in embodiment, if no special instructions, is this area conventional method.
Embodiment 1: the structure of low yield urethanes saccharomyces cerevisiae
Starting strain CGMCC No.3373 used by this example.Described escherichia coli DH5a is purchased from Takara company.Described YEPD culture medium is general complete medium, and solid medium contains 2% import agar powder.
According to the Yeast genome data in Genebank and integrated plasmid sequence, devise following primer.
Primer used in table 1 the present embodiment
Note: underscore part is restriction enzyme site.
Taq archaeal dna polymerase PCR amplification system used in table 2 (a) the present embodiment
PrimeSTARGXLDNA polymerase PCR amplification system used in table 2 (b) the present embodiment
Table 3 (a) Solution I ligase method of attachment
Note: reaction condition is 16C water-bath, overnight connects
Table 3 (b) Infusion ligase method of attachment
Note: reaction condition is 50C water-bath, connects 30min
(1) recombiant plasmid Yep-APD1,2The structure of KB
Recombiant plasmid Yep-APD1,2The structure flow process of KB is as shown in Figure 1；
With plasmid pPGK1 as template, PGK1-U and PGK1-D is primer, Taq archaeal dna polymerase, and PCR expands strong promoter PGK1 gene, PCR reaction condition: 95 DEG C of 5min；94 DEG C of 1min, 54 DEG C of 1min, 72 DEG C of 105s, 30 circulations；72℃10min. With restricted enzyme BamHI and SalI, vector plasmid YEP352 and PGK1 genetic fragment are carried out enzyme action, then use Solution Both are connected structure plasmid Yep-P by I ligase.
With the yeast strain genome that sets out as template, DUR1,2-U and DUR1,2-D is primer, PrimeSTAR GXL DNA Polymerase, PCR expands DUR1,2 genes, PCR reaction condition: 95 DEG C of 5min；98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 335s, 30 are followed Ring；72℃10min.With restricted enzyme XhoI, plasmid Yep-P carried out enzyme action, then with Infusion ligase by DUR1,2 Plasmid Yep-P after genetic fragment and enzyme action connects structure plasmid Yep-PD1,2。
With plasmid pUG6 as template, Kan-U and Kan-D is primer, Taq archaeal dna polymerase, and PCR expands KanMX gene, PCR reaction condition: 95 DEG C of 5min；94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 100s, 30 circulations；72℃10min.In restricted Cut enzyme BamHI to plasmid Yep-PD1,2Carry out enzyme action, then with Infusion ligase by the matter after KanMX genetic fragment and enzyme action Grain Yep-PD1,2Connect and build plasmid Yep-PD1,2K。
With the yeast strain genome that sets out as template, UA-U and UA-D is primer, Taq archaeal dna polymerase, and PCR expands DUR1, the upstream homology arm CUA of 2 genes, PCR reaction condition: 95 DEG C of 5min；94 DEG C of 1min, 53 DEG C of 1min, 72 DEG C of 60s, 30 Circulation；72℃10min.With Restriction enzyme Sma I to plasmid Yep-PD1,2K carries out enzyme action, then will with Infusion ligase DUR1, the plasmid Yep-PD after the upstream homology arm CUA genetic fragment of 2 genes and enzyme action1,2K connects structure plasmid Yep-APD1, 2K。
With the yeast strain genome that sets out as template, UB-U and UB-D is primer, and PCR expands DUR1, and the downstream of 2 genes is same Source arm CUB, PCR reaction condition: 95 DEG C of 5min；94 DEG C of 1min, 53 DEG C of 1min, 72 DEG C of 60s, 30 circulations；72℃10min.With Restricted enzyme SphI is to plasmid Yep-APD1,2K carries out enzyme action, then with Infusion ligase by DUR1, the downstream of 2 genes Plasmid Yep-APD after homology arm CUB genetic fragment and enzyme action1,2K connects structure plasmid Yep-APD1,2KB。
PCR the result is as in figure 2 it is shown, wherein: in (a), M is marker；1 is with Yep-APD1,2KB is template, PGK1- U/PGK1-D is that primer PCR expands PGK1p-DUR1,2-PGK1tFragment；
B in (), M is marker；1 is with Yep-APD1,2KB is template, and CU-U/CU-D is that primer PCR expands CUA- KanMX-PGK1p-DUR1,2-PGK1t-CUB fragment.
(2) DUR1, the process LAN of 2 genes while of knocking out of CAR1 gene
With plasmid Yep-APD1,2KB is template, CU-U and CU-D is primer, PrimeSTAR GXL archaeal dna polymerase, PCR Amplification " CUA-KanMX-PGK1p-DUR1,2-PGK1t-CUB " recombinant fragment, PCR reaction condition: 95 DEG C of 5min；98 DEG C of 10s, 55 DEG C 5s, 72 DEG C of 660s, 30 circulations；72℃10min.
By the method for lithium acetate transformation, PCR primer is imported saccharomyces cerevisiae starting strain and obtain recombinant bacterial strain 1, pass through G418 resistance screening recon carries out PCR checking, at DUR1, the upstream homology arm CUA exterior design primer U-S of 2 genes, KanMX interior sequences design primer KU-S；Downstream homology arm at PGK1 interior sequences design primer PU-X, DUR1,2 gene CUB exterior design primer U-X, is used for verifying CAR1 gene knockout DUR1,2 gene overexpressions simultaneously.With U-S and KU-S for drawing Thing, amplifiable go out size be the fragment of 1208bp；With PD-X and D-X as primer, amplifiable go out size be the fragment of 1336bp, and The fragment that PCR amplifies is in the same size with intended purpose product, and starting strain then can not expand and obtain this fragment, and PCR tests Shown in card result such as Fig. 3 (a).
By the method for lithium acetate transformation the pGAPza plasmid with Cre recombinase turned and obtain in recombinant bacterial strain 1 Recombinant bacterial strain 2；Picking transformant induces 4-5h, dilution spread in galactose culture medium, chooses single bacterium colony on YEPD flat board, Photocopy is in G418 resistant panel again；The bacterial strain choosing growth on YEPD flat board and do not grow in G418 resistant panel, carries Take genome and carry out PCR checking, i.e. with the genome of recombinant bacterial strain 2 for template amplification KanMX fragment, it is impossible to obtain 1600bp left Right band, recombinant bacterial strain 1 then can expand and obtain this fragment, shown in PCR the result such as Fig. 3 (b).By ferment correct for checking Female single bacterium colony is received and is carried out Secondary Culture in YEPD fluid medium, and every 12h transfers once, passes number instead of rear pGAPza plasmid i.e. Can lose, extract yeast plasmid and carry out PCR checking as shown in Fig. 3 (c).
Result shows, the while of achieving the CAR1 gene knocking out a copy in yeast cells, strong promoter PGK1 crosses table Reach DUR1,2 gene complete sequences.
Embodiment 2: low yield EC Wine brewing yeast strain fermenting experiment
(1) recombinant bacterial strain is tested with the vinous fermentation of starting strain
Recombinant bacterial strain and starting strain are carried out vinous fermentation experiment the most simultaneously, fermentation technology route map:
Grape material → clean, dry, destemming → broken → adjust sugar, acid adjustment → add sulfurous acid, sterilizing → connect bacterium → front Ferment → skin slag separation → after fermentation → testing index
Process conditions:
Pol: 20.45Brix；Acidity: pH 3.51；SO2Addition: 80mg/L；Connect bacterium amount: 3%；Primary fermentation temperature and Time: 25 DEG C, 4.5d；After fermentation temperature and time: 20 DEG C, 9d.
Picking one ring yeast cells, accesses in the test tube equipped with 5mL YEPD culture medium, and 30 DEG C, 180rpm cultivates 12h, then It is inoculated into equipped with in the 150mL triangular flask of 45mLYEPD culture medium by 10% inoculum concentration, 30 DEG C, after 180rpm cultivates 12h, presses 3% inoculum concentration is inoculated into Sucus Vitis viniferae fermentation medium, 25 DEG C of standing for fermentation.Weigh 1 time every 12h, when twice weightlessness is less than 0.3g, primary fermentation terminates.Making skin slag separate by filtered through gauze, 20 DEG C of standings carry out after fermentation, and the after fermentation time is primary fermentation Twice.After fermentation ends, fermentation liquid is filtered, obtain dregs of grape wine sample.Measure CO2Accumulation discharge capacity, alcoholic strength, remnants The fermenting property indexs such as reducing sugar, total acid and pH, result such as table 4.
The fermenting property of table 4 parent strain and recombinant bacterial strain measures
Note: shown data are the meansigma methods of three parallel test results.
(2) urea content during wine surveyed by liquid chromatography
1. principle
9-hydroxyl ton and carbamide generate in acid condition and can produce the derivant (N-9H-with fluorescent characteristic Xanthen-9-yl-X), reaction principle as shown in Figure 4, then the separation through performance liquid chromatographic column C18, the inspection of fluorescence detector Survey, use quantified by external standard method.
2. analyte derivative processes
Take 500 μ L wine sample dehydrated alcohol and be diluted to 1000 μ L, then take the wine sample 500 μ L of dilution, add 9-hydroxyl ton solution 500 μ L and hydrochloric acid solution 100 μ L, mixing, room temperature lucifuge derives 30min, after organic filtering with microporous membrane, treats sample introduction analysis.
3. liquid chromatographic detection condition
Chromatographic column: Agilent-C18 chromatographic column: 250mm × 4.6mm, particle diameter 5 μm, column temperature 35 DEG C；Mobile phase A: acetonitrile； Mobile phase B: 0.02mol/L sodium acetate solution, adjusts pH to be 7.2 with 1% (V/V) acetic acid；Flow rate of mobile phase: 1mL/min；Fluorescence is examined Survey device excitation wavelength lambdaex=240nm, emission wavelength lambdaem=308nm, gain 10；Sample size 20uL；Gradient elution, refers to table 5:
Table 5 elution program
Result such as table 6.Result shows, in recombinant bacterial strain 2 the produced wine of fermentation, urea content is 0.93mg/L, relatively goes out Send out bacterial strain and reduce 40.38%.
(3) EC content during combined gas chromatography mass spectrometry surveys wine
1. principle
After sample adds amino propyl formate internal standard, through parlkaline kieselguhr solid-phase extraction column purification and impurity removal, eluting, concentration After, detect with gas chromatograph-mass spectrometer, inner mark method ration.
2. the pretreatment of sample
With solid phase extraction, sample is carried out pretreatment.The wine sample accurately drawing 2mL or the EC prepared mark product, add 20uL, 10.0mg/L internal standard PC uses solution, 0.3g sodium chloride, and after ultrasonic dissolution, mixing, loading is to alkalescence kieselguhr solid phase extraction Take post, under vacuum, make sample solution slowly penetrate into extraction column, stand about 10min.Through 10mL normal hexane drip washing remove impurity After, about carrying out eluting with 1mL/min flow velocity with 15mL, 5% ethyl acetate-ethyl ether solution, eluent is first through equipped with the anhydrous sulfur of 2g The glass funnel dehydration of acid sodium, regathers in 10mL tool plug scale test tube, and ambient temperature using nitrogen is slowly blown to about 0.5mL, By methanol constant volume to 1.0mL, make liquid to be measured, analyze for GC/MS.
3. the testing conditions of GC/MS
Chromatographic condition: CP-WAX capillary tube 50m*0.25mm*0.25 μm, column temperature 50 DEG C keeps 1min, then with 3 DEG C/min's Speed increases to 180 DEG C, keeps 1min, finally increases to 220 DEG C with the speed of 20 DEG C/min, keeps 10min.Carrier gas: He, carrier gas stream Amount: 1mL/min, injection pattern: do not shunt.
Mass Spectrometry Conditions: ion source temperature 250 DEG C, electron energy 70ev, interface temperature 230 DEG C, electron multiplier voltage 1568V, EC detect ion m/z 62,74,89, quota ion m/z 62.PC detects ion m/z 59,62,74, quota ion m/ z 62。
Result such as table 6.Result shows, in recombinant bacterial strain 2 the produced wine of fermentation, EC content is 12.50 μ g/L, relatively goes out Send out bacterial strain and reduce 34.45%.
Table 6 parent strain and the carbamide of recombinant bacterial strain and EC yield
Note: not data be the meansigma methods of three parallel test results.
(4) amino acid content during wine surveyed by liquid chromatography
1. principle
Sample is carried out DNFB column front derivation, makes amino calculate derivative chemical conversion and be conducive to separating, at ultraviolet 360nm, have absorption Compound, through the separation of performance liquid chromatographic column C18, the detection of UV-detector, use quantified by external standard method.
2. analyte derivative processes
Taking sample 20 μ L, add derivative buffer 200 μ L and derivating agent 200 μ L, mixing, 65 DEG C of water-bath lucifuges take after deriving 1h Going out, after solution is cooled to room temperature, molten buffer of reordering to 1200 μ L and mixes, through organic microporous filter membrane mistake after placement 15min After filter, treat sample introduction analysis.
3. liquid chromatographic detection condition
Chromatographic column: Agilent-C18 chromatographic column: 250mm × 4.6mm, particle diameter 5 μm, column temperature 33 DEG C；Mobile phase A: 50% second Nitrile；Mobile phase B: 0.05mol/L sodium acetate solution；Flow rate of mobile phase: 1mL/min；UV-detector wavelength 360nm, gain 10； Sample size 20 μ L；Gradient elution, refers to table 7:
Table 7 elution program
Result such as table 8.Result shows, recombinant bacterial strain 2 conventional amino acid content in produced wine of fermenting relatively is set out bacterium Strain no significant difference.
The conventional amino acid yield (unit mg/L) of table 8 parent strain and recombinant bacterial strain
Note: shown data are the meansigma methods of three parallel test results.
(5) volatile acid, ester and the content of alcohol in gas chromatography determination wine
1. wine sample pretreatment
Accurately measure internal standard liquid (n-butyl acetate) 0.15mL in 10mL volumetric flask, with wine sample constant volume to be measured and mix, After 400 μm aperture membrane filtrations, carry out high resolution gas chromatography mensuration.
2. GC conditions
Gas chromatograph: Agilent 7890C；Chromatographic column: AT.LZP-930,230 DEG C, 50m × 320 μ m 1 μm；Detection Device: fid detector, detector temperature: 200 DEG C；Carrier gas: High Purity Nitrogen, flow velocity 5mL/min；Testing conditions: temperature programming, 50 DEG C Keep 8min, 5 DEG C/min to be raised to 120 DEG C, keep 8min；Injector temperature: 200 DEG C；Sample size: 1.0 μ L；Shunting mode: point Stream, split ratio is 10:1.
Result such as table 9.Result shows, common volatile acid, ester and alcohol content in recombinant bacterial strain 2 the produced wine of fermentation Relatively starting strain no significant difference.
Table 9 parent strain and the volatile acid of recombinant bacterial strain, ester and alcohol yield (unit mg/L)
Note: shown data are the meansigma methods of three parallel test results.

Claims (9)

1. a strain low yield urethanes Wine brewing yeast strain, is the arginine knocking out a copy in yeast starting strain Enzyme gene C AR1, simultaneously process LAN urea groups amidase gene DUR1,2 obtain.
A strain low yield urethanes Wine brewing yeast strain the most as claimed in claim 1, it is characterised in that described in set out bacterium Strain is saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3373.
A strain low yield urethanes Wine brewing yeast strain the most as claimed in claim 1, it is characterised in that described CAR1 base Because its Gene ID is: 855993, nucleotide sequence is as shown in SEQ NO:1 in table；Described DUR1, its Gene ID of 2 genes be: 852507, nucleotide sequence is as shown in SEQ NO:2 in table；Its Gene ID of described promoter PGK1 is: 850370, nucleotides sequence Row are as shown in SEQ ID NO:3 in table.
4. the preparation method of a strain low yield urethanes Wine brewing yeast strain, comprises the following steps:
(1) recombiant plasmid Yep-APD1,2The structure of KB
1. with plasmid pPGK1 as template, PCR expands strong promoter PGK1 gene, with restricted enzyme BamHI and SalI to load Body constitution grain YEP352 and PGK1 genetic fragment carry out enzyme action, then with Solution I ligase, both connect structure plasmid Yep- P；
2. with the yeast strain genome that sets out as template, PCR expands DUR1, and 2 genes, with restricted enzyme XhoI to plasmid Yep-P carries out enzyme action, then with Infusion ligase, DUR1, the plasmid Yep-P after 2 genetic fragments and enzyme action is connected structure matter Grain Yep-PD1,2；
1. with plasmid pUG6 as template, PCR expands KanMX gene, with restricted enzyme BamHI to plasmid Yep-PD1,2Carry out Enzyme action, then with Infusion ligase by the plasmid Yep-PD after KanMX genetic fragment and enzyme action1,2Connect and build plasmid Yep- PD1,2K；
4. with the yeast strain genome that sets out as template, PCR expands DUR1, the upstream homology arm CUA of 2 genes, in restricted Cut enzyme SmaI to plasmid Yep-PD1,2K carries out enzyme action, then with Infusion ligase by DUR1, the upstream homology arm CUA of 2 genes Plasmid Yep-PD after genetic fragment and enzyme action1,2K connects structure plasmid Yep-APD1,2K；
5. with the yeast strain genome that sets out as template, PCR expands DUR1, the downstream homology arm CUB of 2 genes, in restricted Cut enzyme SphI to plasmid Yep-APD1,2K carries out enzyme action, then with Infusion ligase by DUR1, the downstream homology arm of 2 genes Plasmid Yep-APD after CUB genetic fragment and enzyme action1,2K connects structure plasmid Yep-APD1,2KB。
(2) DUR1, the process LAN of 2 genes while of knocking out of CAR1 gene
1. with plasmid Yep-APD1,2KB is template, and PCR expands " CUA-KanMX-PGK1p-DUR1,2-PGK1t-CUB " restructuring sheet Section；
2. being imported by the PCR recombinant fragment of step (2)-1. obtain by lithium acetate transformation method sets out in yeast strain, it is thus achieved that knock out The CAR1 gene of one copy, process LAN DUR1 simultaneously, the recombinant bacterial strain 1 of 2 genes；
3. the KanMX gene in bacterial strain is obtained with pGAP plasmid removing step (2)-2., it is thus achieved that without resistant gene, knock out one The CAR1 gene of copy, process LAN DUR1 simultaneously, the recombinant bacterial strain 2 of 2 genes, passes on the recombinant bacterial strain obtained without pGAP plasmid 2。
The preparation method of a strain low yield urethanes Wine brewing yeast strain the most as claimed in claim 4, it is characterised in that Described starting strain is saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3373.
The preparation method of a strain low yield urethanes Wine brewing yeast strain the most as claimed in claim 4, it is characterised in that The described plasmid containing PGK1 strong promoter is Yep352 plasmid.
The preparation method of a strain low yield urethanes Wine brewing yeast strain the most as claimed in claim 4, it is characterised in that The described carrier with KanMX resistance is Yep352 carrier.
The preparation method of a strain low yield urethanes Wine brewing yeast strain the most as claimed in claim 4, it is characterised in that It is lithium acetate transformation method that described genetic fragment imports the method for yeast strain.
9. low yield urethanes saccharomyces cerevisiae application in Production of Wine as claimed in claim 1.
CN201610020591.8A 2016-01-12 2016-01-12 One strain by knocking out CAR1 process LAN DUR1,2 low yield urethanes Wine brewing yeast strain and construction methods thereof Pending CN106119141A (en)

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