CN110343652B - A low-yielding higher alcohol brewer's yeast strain and construction method thereof - Google Patents

Abstract

The invention discloses a low-yielding higher alcohol brewer's yeast strain and a construction method thereof, belonging to the technical field of biological engineering. The low-yielding higher alcohol S. cerevisiae strain of the present invention is obtained by knocking out the key regulatory factor GAT1 gene sequence of S. cerevisiae nitrogen catabolism inhibition gene transcription. After being fermented in the fermentation medium with wheat as the raw material, the low-yielding higher alcohol brewer's yeast strain reduces the total amount of higher alcohol by 21.42% compared with the parent strain. The strain of the invention has good fermentation performance and growth performance, and does not affect the growth performance or other conditions of the recombinant strain.

Description

Low-yield higher alcohol beer yeast strain and construction method thereof

Technical Field

The invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and particularly relates to a beer yeast strain with low yield of higher alcohol and a construction method thereof.

Background

The flavor substances in beer mainly include higher alcohols, esters, aldehydes, phenols, acids, and dithiones. Among them, higher alcohols are one of important chemical substances that contribute to beer flavor and taste. In documents "formation of higher alcohol in beer production and breeding of low-yield higher alcohol yeast strain" and "research progress for controlling content of higher alcohol in fermented wheat beer" it is pointed out that suitable content of higher alcohol has the effects of making beer taste and fragrance rich and making the body of beer soft and harmonious, but too high content of higher alcohol can cause beer to form foreign flavor, which not only affects drinking taste and flavor quality of beer, but also can produce symptoms such as thirst and headache after drinking, and has obvious side effect on body of drinker, thus being not beneficial to body health of drinker. Therefore, the reduction of the higher alcohol content of the beer is very important to enhance the flavor harmony of the beer. Because the wheat raw material has higher protein content and rich nutrition, belongs to highland barley and no hull, and a good filter layer cannot be formed during filtering, so that the filtering is difficult, the beer is generally produced by fermenting the beer yeast strain fermented on the wheat raw material at higher temperature (the main fermentation temperature is 16-20 ℃). The higher alcohol content of the wheat beer is generally as high as about 300mg/L or even higher, which is more than three times of that of the barley beer, and the side effect after drinking is obvious, which is one of the main reasons for inhibiting the development of the wheat beer. In order to reduce the content of higher alcohol in wheat beer in industry, a wort preparation process route is usually adjusted, and the content of alpha-amino nitrogen is reduced, so that the content of the higher alcohol in the fermented beer is reduced, but the style of the finished beer is influenced; another way is to adjust fermentation parameters, which not only affect the growth and propagation of yeast and reduce the fermentation rate, but also affect the content of flavor substances other than higher alcohols in the finished beer, resulting in a change in beer style.

The article "Function and regulation of yeast genes involved in high alcohol and ester metabolism stimulation" reported that higher alcohol can inhibit nerve center, cause damage to sympathetic nerve and optic nerve, etc., and has stronger anesthetic effect than ethanol, wherein, the toxicity of propanol is 8.5 times that of ethanol, isobutanol is 8 times that of ethanol, and isoamylol is 19 times that of ethanol; meanwhile, the higher alcohol has low decomposition and oxidation speed in a human body and long metabolic retention time; these factors cause that drinking beer with too high content of higher alcohol can cause symptoms of thirst, headache and the like of drinkers, which is also the main reason that drinking beer is drunk slowly and is difficult to sober up after being drunk. The excessive higher alcohol is the main source of the beer foreign flavor, and the excessive n-propanol is like ether odor and has bitter taste; excess butanol can contribute to the fusel oil odor typical of beer, while producing an unpleasant bitter taste; if the amyl alcohol exceeds the standard, putrefactive odor and sweat odor are generated; the higher alcohols and their metabolic derivatives generated by reaction with acetic acid mainly include isoamyl acetate, isobutyl acetate, phenylethyl acetate, etc., and the content and ratio (alcohol ester ratio, higher alcohol to ester content ratio) of esters such as ethyl acetate, ethyl caproate, ethyl caprylate, etc., have extremely important influence and contribution to the flavor of beer.

There are two ways of producing higher alcohols in beer: the metabolic pathways for synthesizing higher alcohols by using beer yeast mainly include two pathways: one is an alpha-keto acid catabolic pathway called the ellichi pathway, i.e., the amino acid catabolic pathway; the other is the alpha-keto acid anabolic pathway, known as the harris pathway, the higher alcohol anabolic pathway. Genetic engineering breeding for regulating the content of higher alcohols reported in the literature almost goes around these two routes. For example, Eden et al found that the knockout of BAT1 and BAT2 genes were both effective in reducing the production of higher alcohols, and Shiyu et al found that the production of n-propanol was significantly reduced in recombinant strains with ILV1 gene deletion. However, most of these reports are constructed lager brewing yeasts and bottom fermenting yeasts, and there are only reports on the modification and breeding of genes related to the higher alcohol metabolism of top fermenting yeasts used in wheat beers. In addition, in modern wheat beer fermentation, many technological methods are used for reducing the content of higher alcohol, for example, Korean dragon reduces the content of higher alcohol by changing the technological parameters such as yeast inoculation amount, fermentation temperature, fermentation pressure and the like in beer fermentation, but in actual production, the difference of regulation and control effects among different batches is large, and the application is not ideal. The basic method for reducing the content of the higher alcohol in the wheat beer is to construct a strain with low yield of the higher alcohol.

Disclosure of Invention

The invention aims to construct a saccharomyces cerevisiae strain with low high alcohol yield by regulating a nitrogen metabolic pathway aiming at saccharomyces cerevisiae used by wheat beer.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the invention provides a saccharomyces cerevisiae strain with low high alcohol yield obtained by knocking out the GAT1 gene complete sequence of coding universal amino acid permease Gap1p in a saccharomyces cerevisiae starting strain.

The GAT1 Gene has Gene ID as follows: 850523, the nucleotide sequence is shown in SEQ NO. 1 in the sequence table.

The GAT1 gene is a key regulatory factor for the transcription of nitrogen catabolism suppressor gene of beer yeast. This nitrogen catabolism inhibition inhibits the uptake of arginine and alanine, and stimulates the consumption of branched and aromatic amino acids. The GAT1 gene encodes a Saccharomyces cerevisiae universal amino acid permease, Gap1p, Gap1p, which is capable of transporting the D-and L-isomers of all the common amino acids in proteins; it is an inducible amino acid permease regulated by the abundance of amino acids.

Preferably, the beer yeast starting strain is beer yeast (Saccharomyces cerevisiae) S17 with the preservation number of No. CICC1929.

The construction method of the saccharomyces cerevisiae strain with low yield of higher alcohol comprises the following steps:

(1) amplifying to obtain upstream and downstream sequences of GAT1 gene by taking the genome of the starting strain as a template;

(2) transforming the DNA molecules of the upstream and downstream sequences of the GAT1 gene and a marker gene KanMX into the starting strain to obtain a recombinant strain;

(3) adopting a Cre-LoxP reporter gene rescue system to remove a KanMX resistance gene in the recombinant strain, and losing the pSH-Zeocin plasmid introduced by the KanMX resistance gene;

(4) repeating said steps (1) - (3) with a diploid industrial strain.

Further, the construction method of the saccharomyces cerevisiae strain with low yield of higher alcohol comprises the following specific steps:

1) taking the genome of the starting strain as a template, and carrying out PCR amplification on upstream and downstream homologous sequences of the GAT1 gene;

2) using the plasmid pUG6 as a template, and carrying out PCR amplification to obtain a PCR product containing a KanMX marker gene;

3) transforming the PCR products obtained in the steps 1) and 2) into the starting strain by a lithium acetate chemical transformation method, screening transformants by using a G418 resistant plate, selecting the transformants growing on the G418 resistant plate for PCR verification, and screening to obtain a positive transformant to obtain a recombinant strain;

4) chemically transforming pSH-Zeocin plasmid into the recombinant strain in the step 3) by using a Cre/loxP reporter gene rescue system through lithium acetate, and obtaining a transformant which is removed with a KanMX resistance marker through PCR verification screening to obtain a genetic engineering strain.

5) Subculturing the genetic engineering strain obtained in the step 4), discarding free pSH-Zeocin plasmids, selecting strains of 4 th to 5 th generations and more than 5 th generations, extracting yeast plasmids from the strains, taking the yeast plasmids as templates, performing PCR amplification by using primers, and verifying whether the pSH-Zeocin plasmids are lost;

6) performing second allele knockout on the strain obtained by screening in the step 5):

introducing the recombinant fragments obtained by the methods of the steps 1) and 2) into the strain obtained in the step 5) by using the method of the step 3), screening to obtain a recombinant strain with a second allele deletion, removing the KanMX resistance gene according to the methods of the steps 4) and 5), and discarding the pSH-Zeocin plasmid.

Further, in the step 2), the plasmid pUG6 is used as a template, and GAT1K-F (shown as SEQ NO: 4) and GAT1K-R (shown as SEQ NO: 5) are used as primer pairs to PCR amplify the loxP-KanMX1-loxP fragment knocked out by one allele of the GAT1 gene.

The recombinant strain can be constructed by the method, and the methods are reported in a plurality of documents, such as: gietz R D, Schiestl R h.high-efficiency layer transformation using the LiAc/SS carrier DNA/PEG method [ J ]. Nature Protocols, 2007, 2 (1): 38-41. Other methods known in the art can also be used to construct genetically mutated yeast strains.

Another object of the present invention is to provide the use of the above-mentioned higher alcohol-producing Saccharomyces cerevisiae strain.

Preferably, the use of the higher alcohol-producing Saccharomyces cerevisiae strain in beer fermentation.

In the present invention, the lager brewing yeast S17 is derived from the following articles: sun Z G, Wang M Q, Wang Y P, et al.identification by compatible transactions for core regulatory genes in a top-influencing layer at differential thermal in a transfer [ J ] 2019.

The pSH-Zeocin plasmid is derived from the article: li W, Chen S J, Wang J H, et al. genetic engineering to organic carbon flux for vacuum high alcohol production by Saccharomyces cerevisiae for Chinese Baijiu transfer [ J ]. Applied Microbiology and Biotechnology,2018 (in page 4 table)

The beer yeast strain with low yield of higher alcohol reduces the total amount of higher alcohol by 21.42 percent compared with a parent strain after being fermented in a fermentation culture medium taking wheat as a raw material under the condition that other fermentation performance is not influenced. The strain has good fermentation performance and growth performance, and the growth performance of the recombinant strain is not influenced or other conditions are not generated.

Has the advantages that:

1. the low-yield higher alcohol beer yeast strain provided by the invention can regulate and control the expression of the GATA family transcription factor Gat1p on the premise of keeping good fermentation performance, thereby achieving the effect of reducing higher alcohol and laying a theoretical foundation for brewing wheat beer with good flavor and unique taste.

2. The yield of the yeast higher alcohol obtained by the breeding of the invention is obviously reduced. After the fermentation of wheat beer, it was concluded that after two allele knock-outs of the GAT1 gene, the total amount of higher alcohols in strain S17-D Δ GAT1-k-p was 232.6mg/L, and the total amount of higher alcohols in parent strain S17 was 296.0 mg/L. The total amount of higher alcohols in the strain after knocking out both alleles of GAT1 was reduced by 21.42% compared to the parent strain.

The strain has good fermentation performance and growth performance, and the growth performance of the recombinant strain is not influenced or other conditions are not generated. In addition, the content of other flavor substances except higher alcohol of the strain is not influenced, and the flavor substances in the beer are well reserved.

Drawings

FIG. 1 is a flow chart of homologous recombination strain construction;

FIG. 2 shows the electrophoresis of GAT1A, GAT1B, loxP-KanMX1-loxP fragments,

wherein M is DL5000 DNA marker; lane 1 shows the PCR amplification results (564bp single band) using S17 genome as template and GAT1A-F and GAT1A-R as primer set; lane 2 shows the PCR amplification results (729bp single band) using S17 genome as template and GAT1B-F and GAT1B-R as primer set; lane 3 is the result of PCR amplification using the plasmid pUG6 genome as template and GAT1K-F and GAT1K-R as primer set (1663bp single band);

FIG. 3 is a validated electrophoretogram of yeast strain S17- Δ GAT1 for successful knock-out of one allele of GAT1,

wherein, the lane M is DL5000 DNA marker; lane 1 shows that the PCR products of the upstream primers GAT1-M1-U and GAT1-M1-D are a specific band with a size of 2225 bp; lane 2 is the PCR product of the downstream primers GAT1-M2-U and GAT1-M2-D, and a specific band of 2887bp in size can be seen by 0.8% agarose gel electrophoresis; lanes 3 and 4 are negative controls, no bands;

FIG. 4 is a verified electrophoresis chart of the S17-delta gat1 strain from which KanMX resistance genes are knocked out,

wherein, lane M is DL5000 DNA marker; lane 1 is a fragment (1613bp single fragment) obtained by PCR amplification using the genome of the recombinant strain S17- Δ gat1 as a template and K-F and K-R as primer pairs; lane 2 is the result of PCR amplification using the S17- Δ gat1-K genome as a template and K-F and K-R as primer pairs;

FIG. 5 is a diagram showing a verified electrophoresis of discarded pSH-Zeocin plasmid from S17-. DELTA.gat-k strain,

wherein, lane M is DL5000 DNA marker; lane 1 is a fragment (1172bp single fragment) obtained by PCR amplification using pSH-Zeocin plasmid as a template and Zn-F and Zn-R as primer pairs; lane 2 is the result of PCR amplification using S17- Δ bio5-k-p genome as template and Zn-F and Zn-R as primer pair;

FIG. 6 shows the electrophoresis of DGAT1A, DGAT1B, and D-loxP-KanMX1-loxP fragments,

wherein, lane M is DL5000 DNA marker; lane 1 shows the PCR amplification results (486bp single band) using S17 genome as template and DGAT1A-F and DGAT1A-R as primer set; lane 2 is the result of PCR amplification using S17 genome of Saccharomyces cerevisiae as template and DGAT1B-F and DGAT1B-R as primer set (410bp single band); lane 3 is the result of PCR amplification using plasmid pUG6 genome as template and DGAT1K-F and DGAT1K-R as primer set (1663bp single band);

FIG. 7 is a validated electrophoretogram of a yeast strain that successfully knocked out both alleles of GAT1,

wherein, lane M is DL5000 DNA marker; lane 1 is a fragment (1349bp single fragment) obtained by PCR amplification using the genome of the recombinant strain D.DELTA.gat 1 as a template and DGAT1-M1-U and DGAT1-M1-D as primer pairs; lane 2 is the result of PCR amplification using the S17- Δ gat1-k genome as template and DGAT1-M1-U and DGAT1-M1-D as primer set; lane 3 is a fragment (1632bp single fragment) obtained by PCR amplification using the genome of recombinant strain S17-D Δ gat1 as a template and DGAT1-M2-U and DGAT1-M2-D as primer pairs; lane 4 is the result of PCR amplification using the S17- Δ gat1-k genome as a template and DGAT1-M2-U and DGAT1-M2-D as primer sets;

FIG. 8 is a verified electrophoresis chart of the S17-D Δ gat1 strain from which the KanMX resistance gene has been knocked out,

wherein, lane M is DL5000 DNA marker; lane 1 is a fragment (1613bp single fragment) obtained by PCR amplification using the genome of the recombinant strain S17-D Δ gat1 as a template and K-F and K-R as primer pairs; lane 2 is the result of PCR amplification using the S17-D Δ gat1-K genome as a template and K-F and K-R as primer pairs;

FIG. 9 is a diagram showing a verified electrophoresis of discarded pSH-Zeocin plasmid from strain S17-D Δ gat1-k,

wherein, lane M is DL5000 DNA marker; lane 1 is a fragment (1172bp single fragment) obtained by PCR amplification using pSH-Zeocin plasmid as a template and Zn-F and Zn-R as primer pairs; lane 2 is the result of PCR amplification using S17-D Δ gat1-k-p genome as template and Zn-F and Zn-R as primer pair;

FIG. 10 is a route diagram of the fermentation process of the recombinant strain.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

The lager brewing yeast used in the invention can be any kind of lager brewing yeast strain.

Example 1: construction of beer yeast engineering strain with low-yield higher alcohol

The beer yeast S17 as the initial strain is used in constructing recombinant gene engineering strain through homologous recombination process.

Based on the yeast genome data and the integration plasmid sequence in Genebank, each primer in the following examples was designed.

TABLE 1 PCR primers used in this example

The main construction process of the strain is as follows (the construction flow chart of the homologous recombination strain is shown in the attached figure 1):

1) amplification of a fragment required for one-allele knock-out of the GAT1 Gene

PCR amplification is carried out by taking the genome of the beer yeast S17 as a template and GAT1A-F and GAT1A-R as primer pairs, wherein the length of the PCR amplification is 564bp and the upstream homologous sequence GAT1A segment of one allelic knockout of the GAT1 gene is obtained; PCR amplification is carried out on a downstream homologous sequence GAT1B fragment which is subjected to allelic knockout of the GAT1 gene by taking the genome of the beer yeast S17 as a template and GAT1B-F and GAT1B-R as primer pairs, wherein the length is 729 bp; a loxP-KanMX1-loxP fragment which is subjected to allelic knockout of the GAT1 gene is amplified by PCR by using plasmid pUG6 as a template and GAT1K-F and GAT1K-R as primer pairs, wherein the length of the loxP-KanMX1-loxP fragment is 1663bp, and the electrophoretograms of the GAT1A, GAT1B and loxP-KanMX1-loxP fragment are shown in figure 2.

2) Construction of an allelic knock-out recombinant yeast strain of the GAT1 Gene

And (3) carrying out PCR purification and recovery on the three amplified fragments, then transforming the three fragments into the beer yeast strain by using a lithium acetate transformation method, and selecting a transformant with better growth on a G418 resistant plate for primary screening.

3) Verification of one allelic knock-out recombinant yeast strain of GAT1 Gene

According to the gene sequences at two ends of the beer yeast recombination site and the inserted homologous recombination sequences, two groups of upstream and downstream primers are respectively designed, namely: GAT1-M1-U, GAT1-M1-D, GAT1-M2-U and GAT1-M2-D, and the genome of the transformant with better growth is used as a template to carry out PCR amplification and verify the transformant. The PCR products were subjected to 0.8% agarose gel electrophoresis, respectively. The upstream verification results in 2225bp band (nucleotide sequence is shown as SEQ NO: 26), the downstream verification results in 2887bp band (nucleotide sequence is shown as SEQ NO: 27), which indicates that three fragments are successfully integrated into S17 of the Saccharomyces cerevisiae strain, namely, one allele of GAT1 in S17 is successfully knocked out and is named as S17-delta GAT1, the verification electrophoresis chart is shown as figure 3, PCR products in lane 2 can be subjected to 0.8% agarose gel electrophoresis to see a specific band with size of 2887bp, and the size of the specific band is consistent with the expected size. Lanes 3 and 4 are negative controls, and no bands are as expected. The three recombinant fragments are successfully homologously recombined into the S17 genome of the beer yeast, the recombination position is also correct, and the S17-delta gat1 strain is successfully constructed. .

4) Knockout of KanMX resistance gene in recombinant strain S17-delta gat1

The pSH-Zeocin plasmid was chemically transformed into a beer yeast strain positive transformant S17- Δ gat1 containing KanMX resistance gene with lithium acetate using Cre/loxP reporter gene rescue system, spread on a YEPD plate containing 100. mu.g/mL Zeocin resistance, and cultured at 30 ℃ for 36h in the dark. And selecting a colony with better growth vigor and larger size, inoculating the colony in a YEPD liquid culture medium, extracting the plasmid by using a yeast plasmid extraction kit, and verifying whether the pSH-Zeocin is successfully introduced by PCR. The recombinant strain into which the pSH-Zeocin plasmid has been successfully introduced is inoculated into galactose induction liquid medium for culture for 4-5h, and then is diluted and spread on a common YEPD plate. Single colonies were picked and spotted on YEPD plates without resistance and then replica-printed onto YEPD media with G418 resistance. The resulting strain was grown on YEPD and not on plates containing G418. Extracting a yeast genome, and obtaining a transformant which is knocked out of a KanMX resistance marker through PCR verification and screening by taking K-F and K-R as primers, wherein the transformant is named as S17-delta gat1-K, a verification electrophoresis chart is shown in figure 4, no band of a 2 lane is consistent with the expectation, the result shows that the KanMX resistance gene is successfully knocked out by S17-delta gat1, and the S17-delta gat-K strain is successfully constructed.

5) Discarding of free pSH-Zeocin plasmid from recombinant strain S17- Δ gat1-k

The recombinant strain S17-delta gat1-k with the KanMX resistance genes removed is inoculated into a test tube filled with a fresh YEPD liquid culture medium, and the transfer is carried out once every 12 hours, wherein the transfer times are generally 7-9 times. Extracting the genome of the recombinant strain S17-delta gap1-k after multiple transfer passages, taking pSH-Zeocin plasmids as positive control, taking Zn-F and Zn-R as primers, carrying out PCR verification on the recombinant strain, comparing with non-passage transformants, obtaining the recombinant strain successfully discarding the pSH-Zeocin plasmids through PCR verification and screening, and naming the recombinant strain as S17-delta gap 1-k-p, wherein a verification electrophoresis chart is shown in figure 5 and is consistent with the expectation, which indicates that the S17-delta bio5-k strain successfully discards the pSH-Zeocin plasmids and the S17-delta bio5-k-p strain is successfully constructed.

6) Amplification of a fragment required for the second allele knock-out of the GAT1 Gene

The genome of the beer yeast S17 is taken as a template, DGAT1A-F and DGAT1A-R are taken as primers, and the upstream homologous sequence DGAT1A segment required by knocking out two alleles of the GAT1 gene is amplified by PCR, and the length is 486 bp. The genome of the beer yeast S17 is used as a template, DGAT1B-F and DGAT1B-R are used as primers, and a downstream homologous sequence DGAT1B fragment required by knocking out two alleles of the GAT1 gene is amplified by PCR, wherein the length of the fragment is 410 bp. The plasmid pUG6 is used as a template, DGAT1K-F and DGAT1K-R are used as primers, a D-loxP-KanMX1-loxP fragment required by knocking out two alleles of the GAT1 gene is amplified by PCR, the length is 1663bp, and the electrophoretograms of the DGAT1A, the DGAT1B and the D-loxP-KanMX1-loxP fragment are shown in the attached figure 6.

7) Construction of recombinant yeast strains with second allele knock-out of GAT1 Gene

The upstream homologous sequence DGAT1A fragment, the downstream homologous sequence DGAT1B fragment and the D-loxP-KanMX1-loxP fragment required for knocking out two alleles of the GAT1 gene are transformed into a recombinant strain S17-delta GAT1-k-p by a lithium acetate chemical transformation method. The better transformants grown on the G418 resistant plates were picked and primary screened.

8) Verification of recombinant yeast strain with second allele knockout of GAT1 Gene

According to the gene sequences at two ends of the yeast recombination site and the inserted homologous recombination sequences, two groups of upstream and downstream primers are respectively designed, namely: DGAT1-M1-U, DGAT1-M1-D, DGAT1-M2-U and DGAT1-M2-D, and performing PCR amplification by taking the genome of a transformant with better growth as a template to verify the transformant. The PCR products were subjected to 0.8% agarose gel electrophoresis, respectively. The upstream verification results in a 1349bp band (the nucleotide sequence is shown as SEQ NO: 28), and the downstream verification results in a 1632bp band (the nucleotide sequence is shown as SEQ NO: 29), which indicates that the three fragments are successfully integrated into the recombinant strain S17-delta gat1-k-p and the integration position is correct. Namely, two alleles of GAT1 in the starting strain S17 were successfully knocked out and named S17-D Δ GAT1, and the electrophoretic pattern of the yeast strain is shown in FIG. 7. The verification result is consistent with the expectation, which indicates that the yeast strain S17-D delta GAT1 with two alleles knocked out of the GAT1 gene is successfully constructed.

9) Deletion of KanMX resistance genes in recombinant strain S17-D delta gat1

Chemically transforming pSH-Zeocin plasmid into beer yeast strain positive transformant S17-D delta gat1 containing KanMX resistance gene by using a Cre/loxP reporter gene rescue system through lithium acetate, and obtaining the transformant which is deleted of the KanMX resistance marker through PCR verification and screening by using K-F and K-R as primers according to the method in the step 4), wherein the transformant is named as S17-D delta gat1-K, the verification electrophoresis diagram of the transformant is shown in FIG. 8, the verification result is consistent with the expectation, and the result shows that the KanMX resistance gene is successfully deleted from S17-D delta gat1 and the S17-D delta gat1-K strain is successfully constructed.

10) Discarding of free pSH-Zeocin plasmid from recombinant strain S17-D Δ gat1-k

Free pSH-Zeocin plasmid was lost by multiple subculture transfers. Extracting the genome of the recombinant strain S17-D delta gap1-k after multiple transfer passages, taking pSH-Zeocin plasmid as a positive control, taking Zn-F and Zn-R as primers, and screening to obtain the recombinant strain which successfully discards the pSH-Zeocin plasmid through PCR verification, wherein the recombinant strain is named as S17-D delta gap 1-k-p, and the verification electrophoresis chart is shown in figure 9 and is consistent with the expectation, which shows that the S17-D delta gap1-k strain successfully discards the pSH-Zeocin plasmid and the S17-D delta gap 1-k-p is successfully constructed.

Example 2: fermentation experiment of recombinant strain S17-D delta gat1-k-p wheat beer

1) Route diagram of fermentation process: reference is made to figure 10.

2) The process conditions are as follows: and (3) crushing conditions: the grinding degree is proper for wheat malt without whole grains, and the grinding degree is not easy to be too fine so as to avoid causing too large filtering pressure; liquefying and saccharifying conditions: adding the pulverized wheat malt into warm water at 30 ℃ according to the ratio of the material to the water of 1:4, fully and uniformly stirring, placing in a constant-temperature water bath kettle, keeping the temperature at 30 ℃ for 30min, heating to 65 ℃ at 2.0 ℃/min, keeping the temperature for 90min, quickly heating to 78 ℃ and keeping the temperature for 10 min. Fully stirring once every 5min in the saccharification process; and (3) filtering conditions: filtering the saccharified wheat wort while the wheat wort is hot, and washing grains with hot water at 75 ℃ for 3 times; boiling conditions: cooking the filtrate in an electromagnetic oven, adding 3 ‰ bitter flos Lupuli (based on malt weight) 40min after boiling for 70 min; cooling conditions: naturally cooling to room temperature; centrifugation conditions: centrifuging at 4000r/min for 5 min; adjusting conditions of wort concentration: adjusting the sugar degree to be 12 degrees P; and (3) sterilization conditions: sterilizing at 115 deg.C for 20 min.

3) Wheat malt: 500 g; 2000mL of water is added; 1.5g of hop; yeast inoculation amount: 10% w/v, standing at 20 ℃ for fermentation.

Performing wheat beer fermentation experiments on the beer yeast starting strain S17 and the breeding strain S17-D delta gat1-k-p according to the fermentation process; oscillating and weighing every 12h during fermentation, and recording weight loss; when the strain is fermented for 96 hours, the weight loss of S17 and the bred strain S17-D delta gat1-k-p is not reduced any more, and the culture is stopped and weighed after the fermentation is finished; and (4) determining the weight loss, alcoholic strength, residual sugar, true fermentation degree and main aroma component content of the fermentation liquor. The fermentation performance is characterized by weight loss, alcoholic strength, residual sugar and true fermentation degree, and the result is shown in table 2; the results of the main aroma content are shown in table 3.

4) GC assay analysis method: distilling the fermentation liquor, and carrying out gas chromatography analysis on the liquor sample, wherein the chromatographic conditions are as follows: capillary chromatographic column LZP-930, 50m × 320 μm × 1.0 μm, carrier gas is nitrogen with purity of 99.99%, and split ratio is 1: 10. The injection port temperature is 200 ℃, the detector temperature is 200 ℃, and the injection amount is 1 mu L. The temperature is raised by adopting a program, the temperature is kept at 50 ℃ for 8min, the temperature is raised by 5 ℃/min, the temperature is raised to 150 ℃, and the temperature is kept for 15 min. To maintain the accuracy of the data, each sample was injected twice and the average was taken. Under the same chromatographic condition, the retention time of the chromatographic peak of the known higher alcohol standard substance is compared with the retention time of the chromatographic peak of the higher alcohol substance in the sample for analysis.

Table 2 shows that: in the wheat beer fermentation experiment, the beer yeast strain obtained by the invention has no obvious change in fermentation performance compared with the original strain. The two allelic knockouts of the GAT1 gene had no effect on fermentation performance of S17.

TABLE 2 fermentation Performance of beer fermentation from wheat feedstock

Note: data shown are the average of the results of three replicates

Table 3 shows that the respective higher alcohols of the strain S17-D.DELTA.gat 1-k-p obtained by the present invention were reduced to different degrees compared to the parent strain S17. From the total amount of higher alcohols, the total amount of higher alcohols of the parent strain S17 was 296.0mg/L, whereas the higher alcohol production amount of the strain obtained by the present invention was 232.6mg/L, which was 21.42% lower than that of the parent strain. The strain obtained by the invention can reduce the content of higher alcohol in the wheat beer to a great extent, and provides a theoretical basis for optimizing the beer taste.

TABLE 3 content of main aroma components (mg/L) of beer fermentation of wheat raw material

The wheat beers brewed from the two strains in this test were subjected to sensory evaluation (the panelists consisted of four beer experts in the laboratory), and table 4 shows: compared with the original strain S17, the taste of the wheat beer prepared by fermenting the double-knock-out GAT1 recombinant strain S17-D delta GAT1-k-p is obviously improved, and the phenomenon of topping after drinking is improved. This shows that the strain obtained by the invention can greatly optimize the beer taste.

TABLE 4 evaluation results of beer fermentation of wheat raw Material

In addition, the contents of ethyl acetate and isoamyl acetate produced by the beer yeast S17 are respectively 27.5mg/L and 5.5mg/L, and the total amount is 33 mg/L. The contents of ethyl acetate and isoamyl acetate of the recombinant strain S17-D delta GAT1-k-p with the GAT1 gene knocked out doubly are similar to those of the original strain S17, and no obvious difference exists. On the other hand, if the content of ester substances can be continuously maintained or increased, the taste of the beer can be further improved.

The recombinant strain S17-D delta gat1-k-p keeps the yield of esters (ethyl acetate and isoamyl acetate) on the basis of reducing higher alcohol, thereby showing that the contents of other flavor substances (such as ester substances) except the higher alcohol in the recombinant strain are not influenced by knocking out genes, and the flavor substances in the beer are well kept.

Sequence listing

<110> Tianjin science and technology university

<120> low-yield higher alcohol beer yeast strain and construction method thereof

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<141> 2019-07-24

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<400> 1

atgcacgttt tctttccttt gcttttccgc ccttcccctg ttctgttcat cgcatgtgca 60

tatatatata tagatatata tatacattgt acacggtgca cggtagtgaa cataactatg 120

agcacgaaca gagtcccgaa cctcgacccg gacttgaatt taaacaaaga aatctgggac 180

ctgtactcga gcgcccagaa aatattgccc gattctaacc gtattttgaa cctttcttgg 240

cgtttgcata accgcacgtc tttccatcga attaaccgca taatgcaaca ttctaactct 300

attatggact tctccgcctc gccctttgcc agcggcgtga acgccgctgg cccaggcaac 360

aacgacctcg atgacaccga tactgataac cagcaattct tcctttcaga catgaacctc 420

aacggatctt ctgtttttga aaatgtgttt gacgacgatg acgatgatga tgacgtggag 480

acgcactcca ttgtgcactc agacctgctc aacgacatgg acagcgcttc ccagcgtgct 540

tcacataatg cttctggttt ccctaatttt ctggacactt cctgctcgtc ctccttcgat 600

gaccacttta ttttcaccaa taacttacca tttttaaata ataatagcat taataataat 660

catagtcata atagtagtca taataataac agtcccagca tcgccaataa tacaaacgca 720

aacacaaaca caaacacaag tgcaagtaca aacaccaata gtcctttact gagaagaaac 780

ccctccccat ctatagtgaa gcctggctcg cgaagaaatt cctccgtgag gaagaagaaa 840

cctgctttga agaagatcaa gtcttccact tctgtgcaat cttcggctac tccgccttcg 900

aacacctcat ccaatccgga tataaaatgc tccaactgca caacctccac cactccgctg 960

tggaggaagg accccaaggg tcttcccctg tgcaatgctt gcggcctctt cctcaagctc 1020

cacggcgtca caaggcctct gtcgttgaag actgacatca ttaagaagag acagaggtcg 1080

tctaccaaga taaacaacaa tataacgccc cctccatcgt cgtctctcaa tccgggagca 1140

gcagggaaaa agaaaaacta tacagcaagt gtggcagcgt ccaagaggaa gaactcactg 1200

aacattgtcg cacctttgaa gtctcaggac atacccattc cgaagattgc ctcaccttcc 1260

atcccacaat acctccgctc taacactcgc caccaccttt cgagttccgt acccatcgag 1320

gcggaaacgt tctccagctt tcggcctgat atgaatatga ctatgaacat gaaccttcac 1380

aacgcctcaa cctcctcctt caacaatgaa gccttctgga agcctttgga ctccgcaata 1440

gatcatcatt ctggagacac aaatccaaac tcaaacatga acaccactcc aaatggcaat 1500

ctgagcctgg attggttgaa tctgaattta tag 1533

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 2

ggtcccgctt tccgattt 18

<210> 3

<211> 48

<212> DNA

<213> Artificial sequence ()

<400> 3

cctgcagcgt acgaagcttc agctggtgcg actcatagtg ctgttatt 48

<210> 4

<211> 48

<212> DNA

<213> Artificial sequence ()

<400> 4

aataacagca ctatgagtcg caccagctga agcttcgtac gctgcagg 48

<210> 5

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 5

aatagatgtt gggtcacagc ataggccact agtggatctg ata 43

<210> 6

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 6

tatcagatcc actagtggcc tatgctgtga cccaacatct att 43

<210> 7

<211> 17

<212> DNA

<213> Artificial sequence ()

<400> 7

tttcagacac gaccaaa 17

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 8

ccctgttctg ttcatcgc 18

<210> 9

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 9

cctgcagcgt acgaagcttc agctggcaca atggagtgcg tct 43

<210> 10

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 10

agacgcactc cattgtgcca gctgaagctt cgtacgctgc agg 43

<210> 11

<211> 45

<212> DNA

<213> Artificial sequence ()

<400> 11

aggtgcgaca atgttcagtg gcataggcca ctagtggatc tgata 45

<210> 12

<211> 45

<212> DNA

<213> Artificial sequence ()

<400> 12

tatcagatcc actagtggcc tatgccactg aacattgtcg cacct 45

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 13

tgaagcggac atggaaag 18

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 14

cgatggaccc gaatctcc 18

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 15

ctcagtggca aatcctaa 18

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 16

ggatttgcca ctgaggtt 18

<210> 17

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 17

gccgaccaag ttaccaga 18

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 18

cgcagcatag tgttagtg 18

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 19

ttccgtcagc cagtttag 18

<210> 20

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 20

gactaaactg gctgacgg 18

<210> 21

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 21

aggtctcggt tgctctta 18

<210> 22

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 22

cagctgaagc ttcgtacgc 19

<210> 23

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 23

gcataggcca ctagtggatc tg 22

<210> 24

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 24

cccacacacc atagcttca 19

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 25

agcttgcaaa ttaaagcctt 20

<210> 26

<211> 2225

<212> DNA

<213> Artificial sequence ()

<400> 26

cgatggaccc gaatctcccc tgttcgtgaa cggctttgat attgtccatg tatgtcttgt 60

catcgggcaa gtcagcggtt agtgggtcct tgatgtagaa agtgtcttgc aggtcacgag 120

caggatgctg ttgtgggacg taaagggcat cgaagttcca gaaacctgtc tcgacgtatt 180

ggttcgaggg catctctgtg aatcccatgg aaaagaaaat ttgtctaaat tcctctctga 240

ctttgtttaa ggggtgaaga gcacctgaag atatttgcac accttgagaa ttgaaattgt 300

aaggcttgaa cttcaagtcc ttgtatgcat tggtggagac catgtcggag gtaagatcgg 360

tttccaattt ggtgaggtcg gtcgagaact ctggcccttt ggtaacgttg aaatctgtga 420

ttttaccttg agcaattaac tttcttttct tcaagtcgtt caaaatcttg gcgtcaatgc 480

tatccagatg cgagttgttc ttgatttgcg ctagaataga ttgcgtttca tcagtaagct 540

catttaaatc ggtattttgc aattttgcgg agagttcaag ctcgtttgag gcgtttttgg 600

cgatccagcc gttcttgaaa gctctagcct gaccgacctt accaacttga ggacccagtt 660

tggacatcac atctttgatt tgaagttgac ccaactcttg gatgagcttg actagtttaa 720

tttcgtacga accttcattc aaaatttgag caccttcttt ggtcaagtca tacgtaaccg 780

tgtcgacctt ggaaaactct aacttgttgt gggctttcaa agagttcaaa gcggaaagaa 840

catcttgaga gccgtgctga gggaaagttg ccagtgtgga cttgatctca tccaattcat 900

ctagtttttt tagaatttct aattggaagt cagacatctt tacgttaggg ggtgagagag 960

ggaggggggt gcctttaatg tatatatacg taagatatat atatatatgt atatatatgg 1020

aaatgtattc acaactttac atgtgcatta accacaagta ctgcgtacgt tcaagattac 1080

agcaatgcgt tttattaatt tttcaagcat ttttcacgta gagaggaaca aagtttactg 1140

aaaagaaaag aggtagagaa aaacagaaaa attttttttt tctgtttttc ctgcctcttt 1200

tctttgtttg attcaatatg gtcgaccggg taaacccctg ataaaacgat accaaagccg 1260

ggtcacctaa cttatggcca aatgcgaccg gtcccgcttt ccgattttag ccggcgaaga 1320

cgtacttggc gccataatca aaacctagct tgcccaatac ttctgagttc tacgtggtgc 1380

aaaaatattt tttttttttt gaaaaaccta ccctatttca ttatagatgc atccatcagt 1440

attacggtgt cctcacacaa ccctgtctct gcacaacgta atacctcctt ttcccgtctg 1500

ctagctctca tttcgcggta atccaacttc aaccagcaac ccggatcttc tatacgcagt 1560

ccggtgtgtg ggtgcatgac tgattggtcc ggccgataac aggtgtgctt gcacccagtg 1620

cccaacgtca acaaagcagg aacaacgggc tgataaggga gaagataaga taagataaga 1680

taacaaatca ttgcgtccga ccacaggccg acacatagca gaacgatgtg aagcagcgca 1740

gcatagtgtt agtgccggtg cagctaccgc tggtattaac agccaccaca atacagagca 1800

acaataataa cagcactatg agtcgcacca gctgaagctt cgtacgctgc aggtcgacaa 1860

cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtcta gagatctgtt 1920

tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca gaataccctc 1980

cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag 2040

cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga 2100

agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc cctcacagac 2160

gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca 2220

ctgag 2225

<210> 27

<211> 2887

<212> DNA

<213> Artificial sequence ()

<400> 27

ggatttgcca ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag 60

ttctcacatc acatccgaac ataaacaacc atgggtaagg aaaagactca cgtttcgagg 120

ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat 180

gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg 240

tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta 300

aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat 360

gatgcatggt tactcaccac tgcgatcccc ggcaaaacag cattccaggt attagaagaa 420

tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat 480

tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg 540

caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc 600

tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca 660

gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata 720

ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta 780

tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt 840

attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa 900

tcagtactga caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt 960

atattgtagt tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg 1020

acatcatctg cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta 1080

tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta acgccgccat ccagtgtcga 1140

aaacgagctc tcgagaaccc ttaatataac ttcgtataat gtatgctata cgaagttatt 1200

aggtgatatc agatccacta gtggcctatg ctgtgaccca acatctattg cggcggtgac 1260

agaatagttg aaaggcgcag ggctcacaca caggaatggc cctcccaaat ttaaaagaga 1320

actaaaccag ttatcctagg caattacttt atttgagtct tatatgacgt cactagaagc 1380

tcagtaagag caaccgagac ctgaacatcc tttttttttt gcttctttat ttggcagcat 1440

ttttcaaaaa taataaaatg gaagccgcga gtacgaacaa tgatgtgttc tgggaatacc 1500

tcgtcaaaac aagacaatgg caaggatttt ctttcatcag gcagaaagat ctggatctga 1560

atggcatcat tttgtgatgt gtaaaagcgg gaccttgtta tttcgacttt ttgcatcatg 1620

ttgatgcaat ttgctacttt tccgacggtg cgctccaacg gatgggtatt tccttaataa 1680

caaggcattt ctctggaagt tggcttactg tttgaaatca cagccggtca caaaataaag 1740

taaaaaaact atctctctcc acaagaagta attacaggtt gtatactaca tatgatcgta 1800

tttctttatg aacactaagg agtttcccgc tgtgtaccgc aatatccaca caaaaggaag 1860

gaagaaactt ctgtggcttg acagataaat aactgcagta gtcggtgcgt actaattgtt 1920

tggtcgtgtc tgaaaaatct tgaattttca gaaaagaata agccccaaat gtcagtgatg 1980

gtagtagcag tactccccta cgattttaga tactttagag agcccacctt cagaatcgga 2040

aggaggataa ttttgtaaag cccttctgtt ttttctcttg cataacttat atttccacat 2100

caaaaagtag tgtgctaaga aaaaggagac gagaaaaagg attacggcac tctctgcatc 2160

tagacatata ccaaaagttg ggtttgctca cgaaaatacc ataattgtgg tgtcaaaaaa 2220

atcctgcctc ataataccac tgcagcaatt gtggatgact aaaaaataac ttgcattcca 2280

cgatgttatt ttactttata aagcacctgc aatttttttt tttgtattaa ctcatcgagt 2340

atgtctgatg tgtaaactga accaggctta atatcgtttc taattcttgt tgtgagaaaa 2400

ctttcctgcc tagtgtattt cgtcagggcg aaccttcgga taggcaccga actccgagat 2460

tcttgctcca atttaagaaa taagcttttt ccgtcccaat acaatagaac attattaaag 2520

ttaataataa aaatggcacg gcaccagttg gaagttaaat cccaacattt gctgcaagaa 2580

tagaaaagaa gtcataacaa atttgcatat tacttacggc ttcaaaaaag caccgaaact 2640

aatcgagaaa gcttataata tcggtataca tttgaatgta ttttaactat ttctatatca 2700

aaaaaaaaaa aaaaaattgt atatttttcg ttattctcta attcgtatca cattttatcc 2760

ctaagggaat ctatctctaa tttgcaatag tgtagatacc gtctgcggat agagcgctag 2820

agatagctgg ctttaatctg ctagagtacc atggaacacc agtgataact ctggtaactt 2880

ggtcggc 2887

<210> 28

<211> 1349

<212> DNA

<213> Artificial sequence ()

<400> 28

cgcagcatag tgttagtgcc ggtgcagcta ccgctggtat taacagccac cacaatacag 60

agcaacaata ataacagcac tatgagtcgc acacttgcgg tgcccggccc agccacatat 120

atataggtgt gtgccactcc cggccccggt attagcatgc acgttttctt tcctttgctt 180

ttccgccctt cccctgttct gttcatcgca tgtgcatata tatatataga tatatatata 240

cattgtacac ggtgcacggt agtgaacata actatgagca cgaacagagt cccgaacctc 300

gacccggact tgaatttaaa caaagaaatc tgggacctgt actcgagcgc ccagaaaata 360

ttgcccgatt ctaaccgtat tttgaacctt tcttggcgtt tgcataaccg cacgtctttc 420

catcgaatta accgcataat gcaacattct aactctatta tggacttctc cgcctcgccc 480

tttgccagcg gcgtgaacgc cgctggccca ggcaacaacg acctcgatga caccgatact 540

gataaccagc aattcttcct ttcagacatg aacctcaacg gatcttctgt ttttgaaaat 600

gtgtttgacg acgatgacga tgatgatgac gtggagacgc actccattgt gccagctgaa 660

gcttcgtacg ctgcaggtcg acaaccctta atataacttc gtataatgta tgctatacga 720

agttattagg tctagagatc tgtttagctt gcctcgtccc cgccgggtca cccggccagc 780

gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga 840

tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata 900

cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag 960

cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga 1020

aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc 1080

ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt aaggaaaaga 1140

ctcacgtttc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat 1200

gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg 1260

atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg 1320

agatggtcag actaaactgg ctgacggaa 1349

<210> 29

<211> 1632

<212> DNA

<213> Artificial sequence ()

<400> 29

gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc 60

ctgatgatgc atggttactc accactgcga tccccggcaa aacagcattc caggtattag 120

aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt 180

tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc 240

aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta 300

atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg 360

attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat 420

taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca 480

tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat 540

atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt 600

tctaatcagt actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt 660

tttttatatt gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg 720

cctcgacatc atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa 780

tcgtatgtga atgctggtcg ctatactgct gtcgattcga tactaacgcc gccatccagt 840

gtcgaaaacg agctctcgag aacccttaat ataacttcgt ataatgtatg ctatacgaag 900

ttattaggtg atatcagatc cactagtggc ctatgccact gaacattgtc gcacctttga 960

agtctcagga catacccatt ccgaagattg cctcaccttc catcccacaa tacctccgct 1020

ctaacactcg ccaccacctt tcgagttccg tacccatcga ggcggaaacg ttctccagct 1080

ttcggcctga tatgaatatg actatgaaca tgaaccttca caacgcctca acctcctcct 1140

tcaacaatga agccttctgg aagcctttgg actccgcaat agatcatcat tctggagaca 1200

caaatccaaa ctcaaacatg aacaccactc caaatggcaa tctgagcctg gattggttga 1260

atctgaattt atagatcccc caaaaaaaaa aaagtactcg cttctttcca tgtccgcttc 1320

atatatatat acacatacta atcaaactct atgtatacat agaataaaaa gaagaacact 1380

atatttattt cataaaaaaa aaaaaaaaat aacaaaaaaa gtgcaacatt tatcaaaagc 1440

tcagtgtgcg ttatgcttcc atgtgaccca acatctattg cggcggtgac agaatagttg 1500

aaaggcgcag ggctcacaca caggaatggc cctcccaaat ttaaaagaga actaaaccag 1560

ttatcctagg caattacttt atttgagtct tatatgacgt cactagaagc tcagtaagag 1620

caaccgagac ct 1632

Claims (2)

1. The application of the beer yeast strain with low higher alcohol production is characterized in that the strain is CICC1929 knockout of GAT1 gene with the nucleotide sequence shown in SEQ NO. 1, and the application is the application of the strain in the preparation of beer through wheat fermentation.

2. The use of claim 1, wherein the GAT1 gene is knocked out by homologous recombination.

Images