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

[0055] Example 1: Construction of low-yield higher alcohol brewer's yeast engineering strains

[0056] The starting strain S. cerevisiae S17 was constructed by homologous recombination to construct recombinant genetically engineered strains.

[0057] According to the yeast genome data in Genebank and the integrated plasmid sequence, the primers in the following examples were designed.

[0058] The PCR primers used in the present embodiment of table 1

[0059]

[0060] The main construction process of the strain is as follows (homologous recombination strain construction flow chart is attached figure 1 shown):

[0061] 1) Amplification of the fragment required for one allelic knockout of the GAT1 gene

[0062] Using the genome of Saccharomyces cerevisiae S17 as a template, using GAT1A-F and GAT1A-R as primers to PCR amplify an allelic knockout upstream homologous sequence GAT1A fragment of the GAT1 gene, the length is 564bp; using the genome of Saccharomyces cerevisiae S17 as a template , using GAT1B-F and GAT1B-R as primers to PCR amplify an allelic knockout downstream homologous sequence GAT1B fragment of GAT1 gene, the length is 729bp; using plasmid pUG6 as template, GAT1K-F and GAT1K-R as primers The loxP-KanMX1-loxP fragment of an allelic knockout of the GAT1 gene was amplified by PCR, with a length of 1663bp. The electrophoresis of GAT1A, GAT1B, and loxP-KanMX1-loxP fragments are shown in the attached figure 2 shown.

[0063] 2) Construction of an allelic knockout recombinant yeast strain of GAT1 gene

[0064] After the amplified three fragments were purified and recovered by PCR, they were transformed into brewer's yeast strains by the lithium acetate transformation method, and the transformants with better growth on the G418 resistance plate were selected for preliminary screening.

[0065] 3) Verification of an allelic knockout recombinant yeast strain of GAT1 gene

[0066] According to the gene sequence at both ends of the Saccharomyces cerevisiae recombination site and the inserted homologous recombination sequence, two sets of upstream and downstream primers were designed respectively: GAT1-M1-U, GAT1-M1-D, GAT1-M2-U, GAT1-M2 -D, using the genome of a well-growing transformant as a template, perform PCR amplification to verify the transformant. The obtained PCR products were subjected to 0.8% agarose gel electrophoresis. Upstream verification obtained a 2225bp band (nucleotide sequence as shown in SEQ NO:26), and downstream verification obtained a 2887bp band (nucleotide sequence as shown in SEQ NO:27), indicating that the three fragments have been successfully integrated into S. cerevisiae In strain S17, that is, one allele of GAT1 in S17 was successfully knocked out, named S17-Δgat1, and the verification electropherogram is attached image 3 As shown, the PCR product of the 2 swimming lanes, after 0.8% agarose gel electrophoresis, can see a specific band with a size of 2887bp, and its size is consistent with the expectation. Lanes 3 and 4 are negative controls, and no bands are as expected. It shows that the three recombination fragments have been successfully homologously recombined into the S17 genome of S. cerevisiae S. cerevisiae, and the recombination position is also correct, and the S17-Δgat1 strain has been successfully constructed. .

[0067] 4) Deletion of the KanMX resistance gene in the recombinant strain S17-Δgat1

[0068]Using the Cre/loxP reporter gene rescue system, chemically transform the pSH-Zeocin plasmid into the positive transformant S17-Δgat1 of the S. cerevisiae strain containing the KanMX resistance gene with lithium acetate, and spread it on a Zeocin-resistant YEPD plate containing 100 μg/mL Incubate at 30°C in the dark for 36 hours. Pick larger colonies with better growth and inoculate them in YEPD liquid medium. After extracting the plasmid with a yeast plasmid extraction kit, verify whether pSH-Zeocin has been introduced successfully by PCR. The recombinant strains that have been successfully introduced into the pSH-Zeocin plasmid were inserted into galactose-induced liquid medium and cultured for 4-5 hours, and then diluted and spread on common YEPD plates. Pick out a single colony and inoculate it on a YEPD plate without resistance, and copy it to a YEPD medium containing G418 resistance. Growth on YEPD but no growth on plates containing G418 was the resulting strain. Extract the yeast genome, and use K-F and K-R as primers to obtain a transformant that removes the KanMX resistance marker through PCR verification and screening, named S17-Δgat1-k, and the verification electrophoresis is attached Figure 4 As shown, there is no band in lane 2, which is consistent with expectations, indicating that the KanMX resistance gene has been successfully deleted in S17-Δgat1, and the S17-Δgat-k strain has been successfully constructed.

[0069] 5) Discarding of the free pSH-Zeocin plasmid in the recombinant strain S17-Δgat1-k

[0070] Inoculate the recombinant strain S17-Δgat1-k that has deleted the KanMX resistance gene into a test tube containing fresh YEPD liquid medium, and transfer once every 12 hours, and the number of transfers is generally 7 to 9 times. The genome of the recombinant strain S17-Δgap1-k after multiple transfers was extracted, and the pSH-Zeocin plasmid was used as a positive control, and Zn-F and Zn-R were used as primers to verify the recombinant strain by PCR. As a control transformant, a recombinant strain that successfully discarded the pSH-Zeocin plasmid was obtained through PCR verification and screening, named S17-Δgat1-k-p, and the verification electrophoresis is attached Figure 5 As shown, it is consistent with the expectation, indicating that the S17-Δbio5-k strain has successfully discarded the pSH-Zeocin plasmid, and the S17-Δbio5-k-p strain has been successfully constructed.

[0071] 6) Amplification of the fragment required for the knockout of the second allele of the GAT1 gene

[0072] Using the genome of Saccharomyces cerevisiae S17 as a template and DGAT1A-F and DGAT1A-R as primers, the upstream homologous sequence DGAT1A fragment required for two allelic knockouts of the GAT1 gene was amplified by PCR, with a length of 486 bp. Using the genome of Saccharomyces cerevisiae S17 as a template and DGAT1B-F and DGAT1B-R as primers, the downstream homologous sequence DGAT1B fragment required for two allelic knockouts of the GAT1 gene was amplified by PCR, with a length of 410 bp. Using plasmid pUG6 as template DGAT1K-F and DGAT1K-R as primers, PCR amplifies the D-loxP-KanMX1-loxP fragment required for two allelic knockouts of the GAT1 gene, with a length of 1663bp, in which DGAT1A, DGAT1B, D-loxP -The electropherogram of the KanMX1-loxP fragment is attached Image 6 shown.

[0073] 7) Construction of the second allele knockout recombinant yeast strain of GAT1 gene

[0074] The upstream homologous sequence DGAT1A fragment, the downstream homologous sequence DGAT1B fragment and the D-loxP-KanMX1-loxP fragment required for two allelic knockouts of the GAT1 gene were transformed into recombinant strain S17-Δgat1-k-p by lithium acetate chemical transformation method . Pick the transformants that grow well on the G418 resistance plate for primary screening.

[0075] 8) Verification of the second allele knockout recombinant yeast strain of GAT1 gene

[0076] According to the gene sequence at both ends of the yeast recombination site and the inserted homologous recombination sequence, two sets of upstream and downstream primers were designed respectively: DGAT1-M1-U, DGAT1-M1-D, DGAT1-M2-U, DGAT1-M2- D, Using the genome of the well-growing transformant as a template, perform PCR amplification to verify the transformant. The obtained PCR products were subjected to 0.8% agarose gel electrophoresis. Upstream verification obtained a 1349bp band (nucleotide sequence as shown in SEQ NO:28), and downstream verification obtained a 1632bp (nucleotide sequence as shown in SEQ NO:29) band, indicating that the three fragments have been successfully integrated into the recombinant strain In S17-Δgat1-k-p, and the integration position is correct. That is, the two alleles of GAT1 in the starting strain S17 were successfully knocked out, named S17-DΔgat1, and the yeast strain verification electrophoresis is shown in the attached Figure 7 shown. The verification results were consistent with expectations, indicating that the yeast strain S17-DΔgat1 with two allelic knockouts of the GAT1 gene was successfully constructed.

[0077] 9) Deletion of the KanMX resistance gene in the recombinant strain S17-DΔgat1

[0078] Using the Cre/loxP reporter gene rescue system, chemically transform the pSH-Zeocin plasmid with lithium acetate into the positive transformant S17-DΔgat1 of S. cerevisiae strain containing the KanMX resistance gene, and use K-F and K-R as primers according to the method in step 4). , through PCR verification and screening to obtain a transformant that deleted the KanMX resistance marker, named S17-DΔgat1-k, and its verification electropherogram is as follows Figure 8 As shown, the verification results were consistent with expectations, indicating that S17-DΔgat1 had successfully deleted the KanMX resistance gene, and the S17-DΔgat1-k strain was successfully constructed.

[0079] 10) Discarding of free pSH-Zeocin plasmid in recombinant strain S17-DΔgat1-k

[0080] The episomal pSH-Zeocin plasmid was lost through multiple transfer subcultures. Extract the genome of the recombinant strain S17-DΔgap1-k after multiple transfers and subcultures, use the pSH-Zeocin plasmid as a positive control, and use Zn-F and Zn-R as primers, pass PCR verification, and successfully discard pSH- The recombinant strain of Zeocin plasmid is named S17-DΔgat1-k-p, and the verification electrophoresis is as follows Figure 9 As shown, it is consistent with the expectation, indicating that the S17-DΔgat1-k strain has successfully discarded the pSH-Zeocin plasmid, and the S17-DΔgat1-k-p was successfully constructed.

[0081] Example 2: Fermentation experiment of recombinant strain S17-DΔgat1-k-p wheat beer

[0082] 1) Fermentation process roadmap: refer to the attached Figure 10.

[0083] 2) Process conditions: crushing conditions: the degree of crushing is suitable for wheat malt without whole grains, and the degree of crushing is not too fine, so as not to cause excessive filtration pressure; liquefaction and saccharification conditions: the crushed wheat malt is mixed with a material-to-water ratio of 1:4 Proportionally add warm water at 30°C, stir well, place in a constant temperature water bath, keep at 30°C for 30 minutes, raise the temperature to 65°C at 2.0°C/min, keep for 90 minutes, rapidly raise the temperature to 78°C, and keep for 10 minutes. Fully stir once every 5 minutes during the saccharification process; filter conditions: filter the wheat wort after saccharification while it is hot, and wash the lees with hot water at 75°C for 3 times; ‰ of bitter hops (based on malt weight), the boiling time is 70min; cooling conditions: natural cooling to room temperature; centrifugation conditions: 4000r/min centrifugation for 5min; wort concentration adjustment conditions: adjust the sugar content to 12°P; sterilization conditions: Sterilize at 115°C for 20 minutes.

[0084] 3) Wheat malt: 500g; add water 2000mL; hops 1.5g; yeast inoculum amount: 10% w/v, stand for fermentation at 20°C.

[0085] According to the above fermentation process, the wheat beer fermentation experiment was carried out on the beer yeast strain S17 and the selected strain S17-DΔgat1-k-p; during the fermentation period, it was oscillated and weighed every 12 hours, and the weight loss was recorded; - The weight loss of k-p no longer decreases, it is considered that the fermentation is over, stop the cultivation, and weigh; measure the weight loss, alcohol content, residual sugar, real fermentation degree and main aroma component content of the fermentation broth. The fermentation performance is characterized by weight loss, alcohol content, residual sugar and true fermentation degree, and the results are shown in Table 2; the results of the main aroma components are shown in Table 3.

[0086] 4) GC determination and analysis method: After the fermentation liquid is distilled, the wine sample is analyzed by gas chromatography. The chromatographic conditions are: capillary chromatographic column LZP-930, 50m×320μm×1.0μm, the carrier gas is nitrogen with a purity of 99.99%, and the split ratio 1:10. The temperature of the injection port was 200°C, the temperature of the detector was 200°C, and the injection volume was 1 μL. Using a temperature program, keep at 50°C for 8 minutes, raise the temperature at 5°C/min, raise the temperature to 150°C, and keep for 15 minutes. In order to maintain the accuracy of the data, each sample was injected twice and the average value 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 higher alcohol material chromatographic peak in the sample for analysis.

[0087] Table 2 shows that: during the wheat beer fermentation experiment, compared with the initial original bacteria, the brewer's yeast strain obtained by the present invention has no obvious change in fermentation performance. The two allelic knockouts of the GAT1 gene had no effect on the fermentation performance of S. cerevisiae S17.

[0088] Table 2 Fermentation properties of wheat raw material beer fermentation

[0089]

[0090] Note: The data shown are the average of the results of three parallel experiments

[0091] Table 3 shows that, compared with the parental strain S17, the higher alcohols of the strain S17-DΔgat1-k-p obtained in the present invention are reduced to varying degrees. From the perspective of the total amount of higher alcohols, the total amount of higher alcohols in the parent strain S17 is 296.0 mg/L, while the production of higher alcohols in the strain obtained in the present invention is 232.6 mg/L, which is 21.42% lower than that in the parent strain. This shows that the bacterial strain obtained by the present invention can reduce the content of higher alcohols in wheat beer to a large extent, and provides a theoretical basis for optimizing the taste of beer.

[0092] Table 3 Contents of main aroma components of wheat raw material beer fermentation (mg/L)

[0093]

[0094]

[0095] Sensory evaluation was carried out for the wheat beer brewed by the two bacterial strains in this test (the judges are composed of four beer experts in this laboratory), and table 4 shows: compared with the original bacterial strain S17, the recombinant bacterial strain of the double knockout GAT1 gene obtained by the present invention The taste of the wheat beer produced by S17-DΔgat1-k-p through top fermentation was significantly improved, and the phenomenon of topping after drinking was improved. This shows that the bacterial strain obtained by the present invention can optimize the taste of beer to a large extent.

[0096] Table 4 Evaluation results of wheat raw beer fermentation

[0097]

[0098]In addition, it was determined that the contents of ethyl acetate and isoamyl acetate produced by brewer's yeast S17 were 27.5 mg/L and 5.5 mg/L respectively, and the total amount was 33 mg/L. The content of ethyl acetate and isoamyl acetate of the recombinant strain S17-DΔgat1-k-p of the double knockout GAT1 gene in the present invention is similar to that of the original strain S17, without significant difference. It should be noted that too high alcohol-to-ester ratio of wheat beer will have a bad influence on the taste of beer. On the one hand, reducing the production of higher alcohols can significantly improve the taste of beer. On this basis, if we can continue to maintain Or increasing the content of esters will further improve the mouthfeel of beer.

[0099] In the present invention, the recombinant bacterial strain S17-DΔgat1-k-p has achieved the production of esters (ethyl acetate, isoamyl acetate) on the basis of reducing higher alcohols, thus indicating that the knock-out of the gene has no effect on the recombination The content of other flavor substances (such as esters) in the strain has an influence except higher alcohols, and the flavor substances in the beer are well preserved.