CN106929438A - One plant height produces the saccharomyces cerevisiae and its construction method of Tetramethylpyrazine - Google Patents
One plant height produces the saccharomyces cerevisiae and its construction method of Tetramethylpyrazine Download PDFInfo
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Abstract
The invention discloses the Wine brewing yeast strain that a plant height produces Tetramethylpyrazine, belong to technical field of bioengineering.The present invention selected one plant it is similar to parent strain fermenting property, but high yield Tetramethylpyrazine Wine brewing yeast strain.In liquor fermentation experiment, compared with parent strain, other fermenting properties of resulting Wine brewing yeast strain are unaffected, being obviously improved for Tetramethylpyrazine content is realized by the raising of precursor substance 3-hydroxy-2-butanone content, the content of its 3-hydroxy-2-butanone is 880.82mg/L, Tetramethylpyrazine content is 55.53mg/L, has respectively reached 10 times and 4 times of starting strain.The Wine brewing yeast strain significantly improves Tetramethylpyrazine yield, meets high requirement of the white wine association area to yeast, and application prospect is extensive.
Description
Technical field：
The invention belongs to technical field of bioengineering, it is related to the breeding of industrial microorganism, especially a kind of high yield tetramethyl
The saccharomyces cerevisiae and its construction method of pyrazine.
Background technology：
In recent years, people not only focus on mouthfeel and quality to fermented food, and the health efficacy to it is also paid special attention to.Through
Research finds all to contain certain Pyrazine material in Chinese tradition fermented food white wine, vinegar, soy sauce, not only with fragrance
Property, while there is expansion of blood vessels, improving the (wine brewing of the functions such as blood circulation, protect liver (preventing damage of the alcohol to stomach lining and liver)
Science and technology, 2013 (9)：1-6), and wherein the content highest of Tetramethylpyrazine.Ground with healthy science in first China white wine within 2006
Beg in meeting, the micro constitutent Tetramethylpyrazine that Mr. Wu Jianfeng et al. just proposes in white wine is the feature of beneficial health
The factor, while they think daily appropriate drinking white spirit, the Tetramethylpyrazine contained in wine just can reach health care and to a certain degree
Therapeutic efficiency (wine brewing, 2006,33 (6)：13-16).Tetramethylpyrazine (Tetramethylpyrazine, TTMP), also known as river
Rhizome of chuanxiong piperazine, as the chief active alkaloid component of Chinese medicine Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong, with treatment cardiovascular and cerebrovascular disease, suppresses platelet set
The pharmacological action of poly- and liver protecting.TTMP has relatively low taste threshold in white wine, other fragrance matters can be risen obvious
Set off superposition by contrast, the fragrance of plentiful white wine, its content in wine body reaches 4-7mg/L, and its steam force down, it is volatile,
Therefore the wine stage is being evaporated, it is possible to distillate to form former wine together with alcohol, it is not necessary to add special processing and extraction step.
Therefore it not only has important contribution to the local flavor of white wine, while also giving the wholesome function of white wine.
It is the important healthy functions factor in white wine that scholar generally accepts TTMP in the industry, and at present on TTMP in white wine
Correlative study, it has also become the important directions that researching white spirit is acted on health.Xu Yan et al. has newly separated one plant of withered grass gemma
Bacillus XZ1124, can be with glucose, sucrose, molasses and beancake powder as substrate, by microbe conversion glucose or sucrose and work
Industry raw material molasses and beancake powder turn into interior source precursor 3-hydroxy-2-butanone, so as to improve the yield of TTMP.Solve microbial fermentation production
Production concentration is low in TTMP, need external source add precursor and the low problem of precursor utilization rate (Chinese patent, 200810235366,
2008-12-8).Zhu Bing peaks et al. are then to have screened a bacillus subtilis and three bacillus licheniformis respectively, are being utilized
In reduced sugar fermentation process, coerced by weak acid and add the strategy of glucose, while controlling two stirrings of fermentation stage
Rotating speed, accumulates and improves the utilization rate (40.3%) of precursor substance 3-hydroxy-2-butanone, so improve TTMP yield (Chinese patent,
2010102338685.5,2010-07-28).Wu Qun et al. is then to have invented one kind with vinasse as raw material, adds bacillus subtilis
Bacterium, then by acidity adjustment and add glucose fermentation accumulate 3-hydroxy-2-butanone, both prevented vinasse pollute environment, improve again
The yield (Chinese patent, 201210089617.7,2012-03-30) of TTMP.Research to TTMP in white wine at present focuses mostly on greatly
In the screening of high yield TTMP bacterial strains, the bacterial strain of screening is mostly bacillus subtilis and bacillus licheniformis, this two classes bacterial strain
All it is aerobic strain, and only just has TTMP yield higher close under conditions of neutrality in pH.And the liquor fermentation mistake of reality
Journey is carried out under anaerobism and highly acidity environment, and this two classes strain stops quickly into its growth after fermentation pit and metabolism,
It is limited by very large its application.Only this two classes strain is fermented outside fermentation pit cultivate one's ability generation it is higher
TTMP yield, thus the general Maotai-flavor liquor and sesame flavor style liquor for being appropriate only for that there is the outer accumulation incubation in cellar for storing things of these methods
The production of wine.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) can under the conditions of anaerobism and oxygen consumption growth and breeding, and detesting
Alcoholic fermentation ability is stronger under the conditions of oxygen, while the characteristics of having acidproof, it is the major function bacterium of all kinds of fermented foods.Wine brewing
In yeast alcoholic fermentation process, the precursor substance 3-hydroxy-2-butanone of TTMP is that the α-acetolactic acid produced by valine metabolic pathway passes through
After oxidative deamination reaction generation biacetyl, then synthesized by diacetyl reductase effect, but the 3-hydroxy-2-butanone of generation can be in 2,3- fourths
Synthesis 2,3-butanediol is reduced under the effect of two alcohol dehydrogenase.Therefore common saccharomyces cerevisiae will not accumulate 3-hydroxy-2-butanone, finally exist
Also TTMP contents higher will not be synthesized in fermented food.The saccharomyces cerevisiae industry of accumulation 3-hydroxy-2-butanone is built using molecular breeding technology
Bacterial strain, you can realize obtaining TTMP yield higher while alcoholic fermentation, the wind to improving the fermented food industries such as white wine
Taste quality and health factor content have double meaning.
The content of the invention：
Present invention solves the technical problem that one of be to provide a plant height produce Tetramethylpyrazine (TTMP) saccharomyces cerevisiae.
The yeast strain that sets out is stored in for saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC32315
Chinese industrial Microbiological Culture Collection administrative center, the public is available commercially.
The engineering bacteria in the case where other fermenting properties are unaffected, recombinant bacterial strain than parent strain 3-hydroxy-2-butanone,
TTMP contents have been respectively increased 912.06%, 314.43%.
The saccharomyces cerevisiae engineered yeast strain specifically can be by the way that in the Wine brewing yeast strain that sets out, knockout encodes 2,3- completely
Butanediol dehydrogenation enzyme gene (BDH1), while overexpression diacetyl reductase gene (BDH2) comes under strong promoter PGK1 effects
Realize.
The BDH1 genes its Gene ID is：851239, SEQ NO in nucleotide sequence such as sequence table：Shown in 1；
The BDH2 genes its Gene ID is：851238, SEQ NO in nucleotide sequence such as sequence table：Shown in 2；
The promoter PGK1 its Gene ID is：850370, SEQ ID NO in nucleotide sequence such as sequence table：Shown in 3；
Above-mentioned insertion or missing etc. can be obtained with conventional knockout technique.The existing many document reports of these methods, such as
Joseph Sambrook's etc.《Molecular Cloning:A Laboratory guide》The second edition, Science Press, 1995.Also can be with known in the art
Other methods build the saccharomycete of gene mutation.Wherein preferably it is by knocking out 2,3-butanediol dehydrogenase gene completely
(BDH1) and insert a complete diacetyl reductase gene (BDH2) and obtain.The sequence is missing from the coding of BDH1 100%
Sequence, while increased a complete BDH2 coded sequence.It is this to encode base by knocking out 2,3-butanediol dehydrogenase completely
Because the bacterial strain that (BDH1) and complete overexpression diacetyl reductase gene (BDH2) are obtained is not likely to produce back mutation, bacterial strain it is steady
The strain stability that the methods such as qualitative Billy's point mutation build is higher, is more beneficial for industrial applications.
The method for building above-mentioned saccharomyces cerevisiae engineered yeast provided by the present invention is that PCR is expanded into the restructuring that plasmid is obtained
Box gene fragment, saccharomyces cerevisiae is inserted into lithium acetate transformation method, obtains the Saccharomyces cerevisiae gene engineering bacteria after homologous recombination
Strain.
Described recombinant plasmid Yep-PB2K, is the 2,3-butanediol dehydrogenase coding base that can completely knock out saccharomyces cerevisiae
Because of one recombinant plasmid of complete diacetyl reductase encoding gene BDH2 of BDH1 and insertion.
Invention also provides one kind dedicated for identification is described can overexpression BDH2 under strong promoter PGK1 effects
The gene order of gene recombination plasmid Yep-PB2K, the gene order is with PK-U (SEQ NO：4) with PK-D (SEQ NO：5) it is
Primer, with the recombinant plasmid as template, the primer sequence of specific fragment amplification is：
PK-U：5’-GCATTTTATCCGTACTCCTG-3’
PK-D：5’-TGTCAATCCTGCCTTCTTCC-3’
Amplified fragments sequencing is a specific sequence, SEQ NO in nucleotide sequence such as core sequence table：Shown in 6.
Invention also provides a kind of gene sequence dedicated for identifying the saccharomyces cerevisiae engineered yeast of the high yield TTMP
Row, the gene order is with one group of B2-B1S-U (SEQ NO：7)、B2-B1S-D(SEQ NO：8) with two groups of B2-B1X-U (SEQ
NO：9)、B2-B1X-D(SEQ NO：10) be primer, the saccharomyces cerevisiae engineered yeast pnca gene group with the high yield TTMP as template,
Enter performing PCR amplification, verify recombinant bacterial strain.Primer sequence is：
One group of sense primer B2-B1S-U：5’-CGATTGCTGATGGTGGAG-3’
One group of anti-sense primer B2-B1S-D：5’-GGTTGTTTATGTTCGGATG-3’
Two groups of sense primer B2-B1X-U：5’-TTCTATGTTCGGGTTCAG-3’
Two groups of anti-sense primer B2-B1X-D：5’-ACGCCATTTATTTCAGGAG-3’
One group of PCR primer through 0.8% agarose gel electrophoresis, it can be seen that the specific band of a treaty 1.2kb, its
Size is suitable with expection；Two groups of PCR primer through 0.8% agarose gel electrophoresis, it can be seen that the specificity of a treaty 1.7kb
Band, its size is suitable with expection, in illustrating that recombination box fragment has successfully recombinated saccharomyces cerevisiae genome, restructuring wine brewing
Yeast gene engineering bacterial strain is successfully constructed.
Advantages and positive effects of the present invention：
One is the yeast 2,3-butanediol dehydrogenase coding base that the recombinant Saccharomyces cerevisiae engineering bacteria that the present invention builds is knocked out
Because 100% missing, the KanMX resistant genes being not likely to produce in back mutation, and recombinant bacterial strain have been knocked out, it is ensured that bacterium
Strain and the security of fermented product；Two is the generation that the recombinant Saccharomyces cerevisiae engineered strain that the present invention builds can improve 3-hydroxy-2-butanone
Amount, and then the growing amount of Tetramethylpyrazine is improved, and other fermenting properties do not have significant change, realize in liquor fermentation process
Middle synchronization carries out producing wine and high yield health factor.
【Brief description of the drawings】：
Fig. 1 is Yep-PB2K plasmid construction processes
Fig. 2 is the regrouping process of recombination box fragment and gene BDH1
The checking of BDH2 gene overexpressions recombinant bacterial strain while Fig. 3 is BDH1 gene knockouts
Fig. 4 is the checking of BDH1 gene delection recombinant bacterial strains
【Specific embodiment】：
Method in following embodiments, unless otherwise instructed, is conventional method.
Embodiment 1：Knock out the structure of BDH1 genes and overexpression BDH2 gene Wine brewing yeast strains
Starting strain used by this example is saccharomyces cerevisiae CICC32315, and the escherichia coli DH5a is public purchased from Takara
Department, the YEPD culture mediums are general complete medium.
The structure flow of recombinant plasmid Yep-PB2K is as shown in Figure 1.By PGK1 promoters and terminator on pPGK1 plasmids
Gene enzyme is connected with Yep352 plasmids and obtains Yep-PGK1 again after scaling off；To be obtained with PCR method from saccharomyces cerevisiae
Coding diacetyl reductase BDH2 genes be inserted between PGK1 promoters and terminator, obtain Yep-PGK1-BDH2；In the future
Come from the connection of the KanMX resistant genes on pUG6 and obtain plasmid Yep-PGK1-BDH2-Kan, be named as plasmid Yep-PB2K.
BDH1 regrouping process is as shown in Figure 2.It is template with the vector plasmid Yep-PB2K for building, using long primer B1C-U
(SEQ NO：11) with B1C-D (SEQ NO：12) expand to obtain with PCR method and carry 2,3-butanediol dehydrogenase BDH1 genes two
The recombination box fragment of end homologous fragment B1A and B1B, the long primer sequence of sequence fragment amplification is：
B1C-U：5’-CGAGGGCAGCAGTTTAACATCAAGCCGGATTTGCTCACGCTACTTTGACCCCTTTTCGTT
TCGACGGAGACAGCTGAAGCTTCGTACGC-3’
B1C-D：5’-TTCTGTCTGTTTCATAACAACAAATATTATAAAAGAAAATACATAATCTTAGATACTACA
AATGAGCCGCTAACGAACGCAGAATTTTC-3’
The recombination box fragment for obtaining will be expanded with lithium acetate transformation method and inserts saccharomyces cerevisiae, by B1A and B1B fragments
With the homologous sequence homologous recombination of BDH1 genes both sides on yeast chromosomal, so as to be incorporated on yeast chromosomal and with chromosome
Replicate together.By G418 resistance screening recons after conversion, PGK1-BDH2-Kan fragments substituted on yeast chromosomal
BDH1 genetic fragments, so as to realize knocking out completely and inserting a complete BDH2 gene for the gene.
Then by the recon obtained by G418 resistance screenings enter performing PCR checking, outside BDH1 upstream region of gene and
KanMX interior sequences design a pair of upstream and downstream fixed point checking primer B2-B1S-U, B2-B1S-D, in PGK1 interior sequences and BDH1
Downstream of gene exterior design a pair of upstream and downstream fixed point checking primer B2-B1X-U, B2-B1X-D, for verifying BDH1 gene knockouts
The success of BDH2 gene overexpression strain constructions simultaneously, purified for two generations.
PCR the results are as shown in figure 3, swimming lane M is marker；It is respectively shown in swimming lane 1,2,3 and is converted with recombinant bacterial strain
Son, a purifying generation, purifying second-generation bacterial pnca gene group are template, with B2-B1S-U and B2-B1S-D as primer, amplify and have come greatly
Small is the fragment of 1162bp, and swimming lane 4 can not then be expanded as template with starting strain genome and obtain the fragment；Swimming lane 5,6,7
It is shown to be respectively with recombinant bacterial strain transformant, a purifying generation, purifying second-generation bacterial pnca gene group as template, with B2-B1X-U and B2-
B1X-D is primer, and it is the fragment of 1696bp to have amplified size next, and swimming lane 8 then can not with starting strain genome as template
Amplification obtains the fragment, therefore checking illustrates that BDH2 gene overexpression recombinant bacterial strains are successfully constructed BDH1 gene knockouts simultaneously, and
Do not undergone mutation by purifying passage, then by the resistant gene KanMX removals in transformant, to obtain applicable wine brewing ferment
Female engineering strain.
Embodiment 2：BDH1 genes lack the structure of Wine brewing yeast strain completely
According to the method in embodiment 1, design following scheme obtains the saccharomyces cerevisiae recombinant bacterial strain for knocking out BDH1 genes, this
Starting strain used by example is also saccharomyces cerevisiae CICC32315, and the YEPD culture mediums are general complete medium.
With the KanMX resistant genes on pUG6 plasmids as template, SEQ NO in nucleotide sequence such as nucleotides sequence list：13
It is shown.Using long primer B1K-U (SEQ NO：14) with B1K-D (SEQ NO：15) PCR method amplification is carried out to obtain with 2,3-
The recombination fragment of butanediol dehydrogenase BDH1 genes two ends homologous fragments B1KA and B1KB, the long primer of sequence fragment amplification
Sequence is：
B1K-U：5’-GGAACTAAAAAAAGTTTTAATTAATTATGAGAGCTTTGGCATATTTCAACAGCTGAAGCT
TCGTACGC-3’
B1K-D：5’-CGCGAGGGGCCCCAAATATTATTTTGTCATTACTTCATTTCACCGTGCATAGGCCACTAG
TGGATCTG-3’
The recombination fragment B1KA-KanMX-B1KB for obtaining will be expanded with lithium acetate transformation method and inserts saccharomyces cerevisiae, led to
The homologous sequence homologous recombination of B1KA and B1KB fragments and BDH1 genes both sides on yeast chromosomal is crossed, so as to be incorporated into yeast dye
Replicated on colour solid and with chromosome.By G418 resistance screening recons after conversion, KanMX genetic fragments substituted for yeast
BDH1 genetic fragments on chromosome, so as to realize the knockout completely of the BDH1 genes.
Then by the recon obtained by G418 resistance screenings enter performing PCR checking, outside BDH1 gene upstream and downstream and
KanMX interior sequences design one group of fixed point checking primer K-B1S-U (SEQ NO：16)、K-B1S-D(SEQ NO：17) with two groups
Fixed point checking primer K-B1X-U (SEQ NO：18)、K-B1X-D(SEQ NO：19), for verifying the success of BDH1 gene knockouts,
Purified for two generations, the primer sequence of sequence fragment checking is：
One group of sense primer K-B1S-U：5’-AAGCCGATAATGAGAAGA-3’
One group of anti-sense primer K-B1S-D：5’-CTGAGCGAGACGAAATAC-3’
One group of sense primer K-B1X-U：5’-AATCAGGTGCGACAATCT-3’
One group of anti-sense primer K-B1X-D：5’-TTCTTGGCTGCTGTTCTA-3’
PCR the results are as shown in figure 4, swimming lane M is marker；It is respectively shown in swimming lane 1,2,3 and is converted with recombinant bacterial strain
Son, a purifying generation, purifying second-generation bacterial pnca gene group are template, with K-B1S-U and K-B1S-D as primer, have amplified size next
It is the fragment of 1473bp, and swimming lane 4 can not then be expanded as template with starting strain genome and obtain the fragment；The institute of swimming lane 5,6,7
Show respectively with recombinant bacterial strain transformant, a purifying generation, purifying second-generation bacterial pnca gene group as template, with K-B1X-U and K-B1X-D
It is primer, it is the fragment of 1461bp to have amplified size next, and swimming lane 8 can not then be expanded by template of starting strain genome
Successfully constructed to the fragment, therefore checking explanation BDH1 gene delection recombinant bacterial strains, and do not undergone mutation by purifying passage,
The resistant gene KanMX in transformant is removed again, to obtain applicable Saccharomyces cerevisiae gene engineering bacteria strain.
Embodiment 3：Knock out BDH1 genes and overexpression BDH2 gene Wine brewing yeast strain fermenting experiments
(1) recombinant bacterial strain is tested with the corn thick mash fermentation of starting strain
Recombinant bacterial strain and starting strain are carried out into corn thick mash fermentation experiment simultaneously respectively, zymotechnique route is：Corn
Powder → soak → liquefy → be saccharified → cool down → connect bacterium → fermentation → steaming wine → testing index；
Process conditions：
Soaking conditionses：60~70 DEG C, impregnate 20min；Liquefaction condition：85~90 DEG C, add Thermostable α-Amylase, liquefaction
90min；Saccharification condition：55~60 DEG C, carbohydrase is added, be saccharified 20min.
Bacterial strain CO is determined after fermentation ends2The fermenting property indexs such as accumulation discharge capacity, alcoholic strength and residual reduced sugar, as a result such as
Table 1, alcohol content and residual sugar the content no significant difference compared with starting strain after recombinant bacterial strain fermentation, clpp gene in this example
Except will not have a negative impact to the basic fermenting property of bacterial strain with the operation of overexpression.
The fermenting property of the parent strain of table 1 and recombinant bacterial strain is determined
Note：Shown data are three average values of parallel test result.
(2) measure of 3-hydroxy-2-butanone and Tetramethylpyrazine (TTMP) yield
As shown in Table 2, the content of the 3-hydroxy-2-butanone of recombinant bacterial strain, 2,3-butanediol and Tetramethylpyrazine (TTMP) is respectively
880.82mg/L, 236.95mg/L and 55.53mg/L, 3-hydroxy-2-butanone, TTMP than starting strain be respectively increased 912.06%,
314.43%, the growing amount of 2,3-butanediol reduces about 68.11%.Therefore from the results, it was seen that BDH1 genes lack completely
The recombinant bacterial strain of overexpression BDH2 genes enhances biacetyl and arrives 3-hydroxy-2-butanone under strong promoter PGK1 effects while mistake
Convert and reduce 3-hydroxy-2-butanone to the conversion of 2,3-butanediol so that the yield of 3-hydroxy-2-butanone is largely accumulated, and then improves tetramethyl
The yield of pyrazine (TTMP).
The parent strain of table 2 and recombinant bacterial strain primary product growing amount
Note：Shown data are three average values of parallel test result.
Embodiment 4：BDH1 genes lack Wine brewing yeast strain fermenting experiment completely
Recombinant bacterial strain and starting strain are carried out into corn thick mash fermentation experiment simultaneously respectively according to the method for embodiment 3, is surveyed
Fermenting property index and principal product growing amount as shown in following two tables.
As shown in Table 3, the alcohol content and residual sugar content after recombinant bacterial strain fermentation are no substantially poor compared with starting strain
Not, illustrating the operation of gene knockout in this example will not have a negative impact to the basic fermenting property of bacterial strain.
The fermenting property of the parent strain of table 3 and recombinant bacterial strain is determined
Note：Shown data are three average values of parallel test result.
As shown in Table 4, the content of the 3-hydroxy-2-butanone of recombinant bacterial strain, 2,3-butanediol and Tetramethylpyrazine (TTMP) is respectively than going out
Hair bacterial strain improves 594.91%, 188.75%, and the growing amount of 2,3-butanediol reduces about 67.23%.Therefore can from result
To find out, BDH1 genes lack reduce 3-hydroxy-2-butanone to the conversion of 2,3-butanediol so that the yield of 3-hydroxy-2-butanone is largely accumulated completely
It is tired, and then improve the yield of Tetramethylpyrazine (TTMP).
The parent strain of table 4 and recombinant bacterial strain primary product growing amount
Note：Shown data are three average values of parallel test result.
Claims (7)
1. a plant height produces the Wine brewing yeast strain of Tetramethylpyrazine, and the starting strain is saccharomyces cerevisiae (Saccharomyces
cerevisiae)CICC32315。
2. a plant height according to claim 1 produces the Wine brewing yeast strain of Tetramethylpyrazine, it is characterised in that in other hairs
In the case that ferment performance is unaffected, recombinant bacterial strain is respectively increased than the 3-hydroxy-2-butanone of parent strain, Tetramethylpyrazine content
912.06%th, 314.43%.
3. a plant height produces the construction method of the Wine brewing yeast strain of Tetramethylpyrazine, it is characterised in that be by building restructuring matter
Grain Yep-PB2K, then by lithium acetate transformation method, at starting strain saccharomyces cerevisiae (Saccharomyces cerevisiae)
In by homologous recombination lack saccharomyces cerevisiae in partial or complete 2,3-butanediol dehydrogenase gene BDH1 sequences, while strong
Promoter PGK1 acts on lower overexpression diacetyl reductase gene BDH2 sequences to realize.
4. a plant height as claimed in claim 3 produces the Wine brewing yeast strain of Tetramethylpyrazine, it is characterised in that the BDH1 bases
Because its Gene ID is：851239, SEQ NO in nucleotide sequence such as table：Shown in 1；The BDH2 genes its Gene ID is：
851238, SEQ NO in nucleotide sequence such as table：Shown in 2；The promoter PGK1 its Gene ID is：850370, nucleotides sequence
SEQ ID NO in row such as sequence table：Shown in 3.
5. method according to claim 3, it is characterised in that be preferably by knocking out saccharomyces cerevisiae completely
2,3-butanediol dehydrogenase coding genes and the complete biacetyl of overexpression in (Saccharomyces cerevisiae) are also
Protoenzyme encoding gene, obtains the Wine brewing yeast strain of the high yield Tetramethylpyrazine described in claim 1, and the bacterial strain is missing from BDH1
100% coded sequence.
6. it is a kind of dedicated for identification claim 3 described in build high yield Tetramethylpyrazine Wine brewing yeast strain recombinant plasmid
The gene order of Yep-PB2K, the gene order is with PK-U and PK-D as primer, with the recombinant plasmid Yep-PB2K genes
Group is template, and amplified fragments sequencing is a specific sequence, as shown in sequence table 5.
7. application of the Wine brewing yeast strain of high yield Tetramethylpyrazine as claimed in claim 1 in liquor production.
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| CN115747085A (en) * | 2022-11-09 | 2023-03-07 | 北京工商大学 | Saccharomycetes kazakhstan yeast for producing various pyrazines and application thereof |
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