A licorice-derived gene element that enhances the tolerance of brewer's yeast and its application

Abstract

This invention discloses a licorice-derived gene element for enhancing the tolerance of Saccharomyces cerevisiae and its application. It establishes a link between the cell wall of Saccharomyces cerevisiae and the licorice-derived gene. Furthermore, it elucidates the regulatory mechanism and function of EC11 in cell wall remodeling. By introducing this gene into yeast cells, it is demonstrated at the metabolite level that EC11 can enhance the yeast's adaptability to high temperature and acetic acid, improve the fermentation efficiency of Saccharomyces cerevisiae in the production process, and increase the yield of bioethanol.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to a licorice-derived gene element for enhancing the tolerance of brewer's yeast and its application. Background Technology

[0002] Saccharomyces cerevisiae, as a microbial chassis, is highly applicable in bioethanol production and the utilization of low-cost raw materials. However, the variable culture environment during fermentation, such as high temperature, high osmotic pressure, and high or low pH concentrations, can inhibit the vitality and metabolic level of Saccharomyces cerevisiae cells. Therefore, modifying the production strain to improve the tolerance of yeast cells to high-temperature acetic acid stress will provide an efficient means for ethanol production.

[0003] Currently, the fermentation industry improves the tolerance and ethanol yield of Saccharomyces cerevisiae through laboratory training, transcription factor engineering, and organelle engineering. For laboratory training, mutant strains rarely exhibit mutations, and most mutations are harmful or neutral. The training process is lengthy, cumbersome, and labor-intensive. Furthermore, rational modification strategies are limited by tolerance mechanisms, lacking universality and resulting in limited improvements. Saccharomyces cerevisiae possesses complex transcriptional regulatory mechanisms to cope with various environmental stressors. Studies have shown that some stress-induced transcription factors have significant effects on improving strain tolerance, such as the acid-responsive transcription factor Haa1 and the osmotic pressure-responsive transcription factor Hot1. Although transcription factor engineering is effective in improving yeast tolerance, this method remains time-consuming, requiring the construction of random mutant libraries and high-throughput screening, limiting its application scope, and its complex mechanisms of action and unpredictable results.

[0004] In addition, overexpression of endogenous or exogenous stress-related genes in yeast cells can enhance the stress tolerance of host cells. In order to cope with stress, signaling pathways can be activated to affect various organelles in Saccharomyces cerevisiae cells. However, for each engineering pathway, target organelles need to be carefully selected. Otherwise, unfavorable physiological environment and insufficient supply of essential cofactors or prerequisites may reduce enzyme activity.

[0005] As the foundation of cell growth, the cell wall in yeast cells undergoes structural changes under various stresses, leading to cell wall stress. Therefore, it is essential to continuously monitor the state and integrity of the cell wall. Research on cell wall-related gene modification is a strategy to improve the tolerance of *Saccharomyces cerevisiae*. Over the past few decades, many cell wall genes associated with *Saccharomyces cerevisiae*'s tolerance to ethanol, heat, or osmotic stress have been reported, but studies on the association between these mechanisms of heat tolerance and acetic acid tolerance and the *Saccharomyces cerevisiae* cell wall are relatively few. Plant cell wall remodeling is an important process in plant response to stress, which offers valuable insights for yeast cell wall remodeling. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide an application of the licorice-derived gene element EC11 in enhancing the tolerance of Saccharomyces cerevisiae.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the application of a licorice-derived gene element EC11 in enhancing the tolerance of Saccharomyces cerevisiae, wherein: the nucleotide sequence of the licorice-derived gene element EC11 is SEQ ID NO.1.

[0010] Another objective of this invention is to overcome the shortcomings of the prior art and provide an EC11 expression cassette containing the licorice-derived gene element EC11.

[0011] As a preferred embodiment of the EC11 expression cassette of the present invention, the EC11 expression cassette includes: the promoter is TDH3 promoter, the terminator is CYC1, and it is labeled with ampicillin antibiotic.

[0012] As a preferred embodiment of the EC11 expression cassette of the present invention, the integration site of the EC11 expression cassette with the yeast genome is HO.

[0013] Another object of the present invention is to overcome the shortcomings of the prior art and provide a receptor cell comprising the EC11 expression cassette.

[0014] As a preferred embodiment of the recipient cell described in this invention, the method includes: introducing the EC11 expression cassette into the recipient cell by electroporation, wherein the recipient cell is Saccharomyces cerevisiae.

[0015] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for improving the tolerance of Saccharomyces cerevisiae, comprising overexpressing the licorice-derived gene element EC11.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of the licorice-derived gene element EC11 in the cell wall remodeling of Saccharomyces cerevisiae.

[0017] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of the licorice-derived gene element EC11 in yeast adaptation to external environmental stress.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of the licorice-derived gene element EC11 in the fermentation of Saccharomyces cerevisiae.

[0019] The beneficial effects of this invention are:

[0020] 1. This invention successfully identified the licorice-derived gene EC11 by mining transcriptome data of licorice root genes related to cell wall under different temperature stresses. A link was established between the cell wall of *Saccharomyces cerevisiae* and the licorice-derived gene. Furthermore, the mechanism and function of EC11 in regulating cell wall remodeling were elucidated. Finally, this gene was introduced into yeast cells, demonstrating at the metabolite level that EC11 can enhance yeast's adaptability to high temperatures and acetic acid, improve the fermentation efficiency of *Saccharomyces cerevisiae* during production, and increase bioethanol production. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0022] Figure 1 This is a volcano diagram of differentially expressed genes in Glycyrrhiza glabra from September to June in Example 1.

[0023] Figure 2 This is a gene classification analysis diagram of September-vs-June in Glycyrrhiza glabra in Example 1.

[0024] Figure 3 This study presents the KEGG enrichment analysis of differentially expressed genes in Glycyrrhiza glabra from September to June in Example 1.

[0025] Figure 4 This shows the growth status of yeast cells overexpressing EC11 under high temperature and acetic acid stress in Example 3.

[0026] Figure 5 The expression level of the EC11 gene under different stress conditions in Example 4 is shown.

[0027] Figure 6 The effect of Saccharomyces cerevisiae overexpressing EC11 on bioethanol production in Example 5. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0029] Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Furthermore, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that excludes other embodiments.

[0030] The raw materials used in this invention are sourced from:

[0031]

[0032]

[0033]

[0034] Instruments used in the embodiments of this invention:

[0035]

[0036]

[0037] Unless otherwise stated, the experimental methods disclosed in this invention all employ conventional techniques in molecular biology and related fields.

[0038] Example 1: Screening for Glycyrrhiza-derived genes with high-temperature acetic acid tolerance

[0039] Differential analysis was performed on the transcriptome data of cell wall-related genes in licorice root under different temperature stresses. Genes with a 2-fold or greater difference in FPKM values ​​and a t-test p-value less than 0.05 were considered significantly differentially expressed genes, including 2659 DEGs with significantly lower expression levels and 1653 DEGs with significantly higher expression levels. Gene enrichment analysis and functional enrichment of these genes were performed based on molecular biological functions, such as... Figures 1-3 In conjunction with the SGD database and literature, nine cell wall-related genes with expression levels differing by more than 2 were identified: EC11 (SEQ ID NO.1), UCBGI (SEQ ID NO.2), EC12 (SEQ ID NO.3), EC23 (SEQ ID NO.4), PSMD11 (SEQ ID NO.5), ER35 (SEQ ID NO.6), ER96 (SEQ ID NO.7), ER28 (SEQ ID NO.8), and ER93 (SEQ ID NO.9).

[0040] Example 2: Construction of different Saccharomyces cerevisiae cells

[0041] Using the wild-type Saccharomyces cerevisiae genome as a template, HOL (left arm of the HO site), TDH3p (TDH3 promoter), licorice-derived genes (EC11, UCBGI, EC12, EC23, PSMD11, ER35, ER96, ER28, ER93), CYC1t (CYC1 terminator) and HOR (right arm of the HO site) were amplified, with KanMX4 (G418 expression cassette) as a selection marker.

[0042] After PCR amplification and agarose gel electrophoresis to verify the correct fragment size, the correct fragments were recovered from the gel. The recovered licorice-derived gene fragments (EC11, UCBGI, EC12, EC23, PSMD11, ER35, ER96, ER28, ER93, CYC1t) and HOR-TDH3p gene fragments were ligated and amplified again using OE-PCR. The HOL-TDH3p-EC11 and other licorice-derived gene fragments (UCBGI, EC12, EC23, PSMD11, ER35, ER96, ER28, ER93, CYC1t) were introduced into BY4742 using electroporation. The purified 700 ng concentration fragments were then transformed into yeast using electroporation. The fragments were homologously integrated with the HO site in the yeast genome using HOL / HOR. Positive transformants were screened using YPDS plates containing G418 and verified by genomic PCR. Single-copy strains were selected from the positive clones.

[0043] The PCR amplification reaction procedure is shown in Table 1, the reaction system is shown in Table 2, and the primer sequences involved are shown in Table 3.

[0044] Table 1

[0045]

[0046] Table 2

[0047]

[0048]

[0049] Table 3

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] Example 3: Determination of yeast cell growth under high temperature and acetic acid stress

[0056] Single clones prepared in Example 2 were inoculated into 2 mL of YPD liquid medium and cultured overnight at 30°C and 200 rpm to obtain activated primary Saccharomyces cerevisiae seed culture. The activated primary Saccharomyces cerevisiae seed culture was transferred at a 10% inoculation rate to test tubes containing YPD liquid medium and cultured at 30°C and 200 rpm for 12-16 h to ensure most yeast cells were in the logarithmic growth phase, yielding secondary Saccharomyces cerevisiae seed culture for shake-flask fermentation. Then, an initial inoculation of OD600 = 0.1 was added to narrow-mouthed conical flasks, and the flasks were sealed with silicone stoppers to maintain microaerobic fermentation conditions. OD600 was measured every 4 h for the first 12 h, and finally at 24 h, 36 h, and 48 h.

[0057] When grown at 30℃, the four strains TDH3p-ER93, TDH3p-FATB, TDH3p-UCBGI, and TDH3p-EC11 all showed better growth than the control group BY4742. However, at a high temperature of 37℃, the growth of all strains was significantly inhibited. The growth of the TDH3p-EC11 strain was better than that of the control group BY4742 and other modified yeast cells. Compared with the 30℃ environment, the OD600 of the TDH3p-EC11 strain increased by 34.2%.

[0058] The results are as follows Figure 4 As shown, when the temperature was raised to 37℃, the growth rate of the EC11 overexpression strain was faster than that of the control strain BY4742. At the same time, after the addition of 0.3% acetic acid and fermentation for 36 h, the OD600 increased by 85%.

[0059] Example 4: Transcriptional Levels of EC11

[0060] Activated Saccharomyces cerevisiae strain EC11 and control strain BY4742 were cultured at 30℃, 37℃, and normal temperature under acetic acid stress for 12 h. Cells were collected, RNA was extracted, and reverse transcription and qRT-PCR were performed. The experimental results are as follows: Figure 5As shown, the transcriptional level of EC11 strain integrated into the BY4742 chassis strain via homologous recombination varied with different fermentation conditions. For example, compared to 30℃, acetic acid, high temperature, and high-temperature acetic acid all stimulated EC11 transcription, increasing its transcriptional level. The effect of acetic acid stress was the most significant, increasing it by 105%. At high temperature, the transcriptional level of EC11 increased by 103%. Under the dual stress of high-temperature acetic acid, it decreased compared to high temperature. High temperature, acetic acid, and high-temperature acetic acid stress conditions can promote the expression of EC11 in yeast.

[0061] Example 5: Effect of Saccharomyces cerevisiae overexpressing EC11 on bioethanol production

[0062] Take 1 ml of Saccharomyces cerevisiae fermentation broth, centrifuge at 12000 rpm for 1 min, accurately pipette 100 μL of the supernatant and transfer it to a centrifuge tube containing 900 μL of distilled water for dilution, and vortex to mix. Transfer the mixed solution through a 0.22 μm aqueous filter membrane to an HPLC vial to remove small solid particles that could clog the HPLC tubing and column. The concentrations of glucose, xylose, and ethanol in the culture medium were determined by HPLC and a refractive index detector. Separation was performed using an Aminex HPX-87H column at 55 °C. The eluent was 5 mM H₂SO₄, and the flow rate was 0.55 mL / min. The ethanol yield was characterized by the formula: ethanol (g) / [consumed 0.51% glucose (g)] × 100%.

[0063] The results are as follows Figure 6 The results showed that the EC11 overexpression strain exhibited a significant difference in ethanol production compared to the control under high-temperature acetic acid stress, with a maximum ethanol yield of 25.3 g / L, representing a 141% increase. Under the dual stress of high temperature and acetic acid, EC11 improved glucose utilization efficiency, ethanol production rate, enhanced the metabolic pathway of the byproduct acetic acid, and reduced acetic acid production, indicating that EC11 is more responsive to the dual stress of high temperature and acetic acid.

[0064] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0065] synthetic construct

[0066] sequence list

[0067] SEQ ID No.1

[0068] Artificial sequence

[0069] SEQ ID NO.1

[0070] ATGGATTCTCAACAAATATCTCTGTTAGACAACCATAAAAAATCGACCCT

[0071] TTCCAAAACTCTCTGGCTACTCCTCTCTGTAGCTGCTGTGGTAAGCTCAAC

[0072] AGCCCTCATTGCTTCTTATCTCATCGACCACTCCTCACCTCACCATTTCTG

[0073] TGAACATGCACTTGATACCACATCCTGCTCAGCCCATGTCTCAGAAGTAG

[0074] CTCAAGTTCCAATCTTGACCACCACCAGAAAAGACCACAAGTTCAAATTG

[0075] CTCCAATCCTTCCTAATGAAGTCCACCTCACCCATTCAGAAAGCCATGGA

[0076] AACAGCCAACGCCATCAAACTCAAGGTTAACAACAATAATCCGAGGGAG

[0077] GAAGCATCTTTGCGGGACTGTGAGCAGCTGATGGACTTGTCCATGGACAG

[0078] AGTTTGGGACTCGGTGATGGCTCTAACAAAAGAGAGTACCAATATTGATT

[0079] CACTGCAAGATGCACACACATGGCTAAGCAGCGTGCTCACTAACCATGCC

[0080] ACTTGCTTGGATGGATTAGATGGGCCCCCTCGGGCCCTGATGGAGGCCCA

[0081] GCTTGAGGACTTGATATCCAGAGCAAGAACCTCTCTGGCCTTGCTTGTTG

[0082] CTGTTTTGCCTCCAAATATCAATCCAAAGGAACTAAATGGAGTAAA

[0083] CAAGTGATTGATGAGGCACTGAATGGGGACTTTTTTCCCTCGTGGGTGAC

[0084] GAGCAAGGAGAGGAGCTTTTGGAGTCTTCAGCTGAGGAGATCAAGCCC

[0085] AATGTTGTGGTGGCTAAGGATGGGAGTGGCAGGTTCAAGACCGTTGCTG

[0086] AGGCTGTGGCATCCGCACCCGAACAGTACAAGGTATGTTATCCA

[0087] AGTGAAAGGGAACGTACAAAGAGAATGTTGAGATTGGAAGAAGAA

[0088] GAAGAATGTGATGCTCGTGGGGGAGGGTATGGATTCAACTGTAATCACT

[0089] GGCAGCTTGAATTATGTCGATAATACTAGCACCTTTCATTCTGCCACTGTT

[0090] GCTGCCGTTGGGGATGGGTTTATAGCCCAGGACATGTGCTTCCAAAATAC

[0091] AGCAGGCCCACAGAAGCACCAGGCCGTGGCCCTCCGCGTCGGTGCCGAC

[0092] CAGTCCGTCATTAACCGCTGCCGCATTGACGCCTATCAAGATACTCTCTA

[0093] CGCTCACTCCAACAGGCAGTTCTACCGTGACTCCTTCATCACCGGCACCG

[0094] TTGATTTCATCTTCGGTAACGCCGCCGTAGTATTCCAGAAGTGCAAGTTG

[0095] GTGGCCCGAAAGCCCATGGATAAGCAGAAGAACATGGTCACGGCCCAAG

[0096] GACGAACAGATCCAAACCAGAACACGGGGACCTCAATCCAGCAATGCGA

[0097] CGTAATACCAAGCTCGGATCTTAAGCCCGTAGTGGGCTCCATCAAATCTT

[0098] ATTTGGGCCGTCCGTGGAAGCCATACTCCAGGACCGTTGTGATGCAGTCC

[0099] TATGTGGACAGCCATATAGACCCAGCAGGGTGGGCCGAATGGGATGACC

[0100] AACACAAGGACTATTTGAAGACATTGTATTATGGGGAGTACATGAACAA

[0101] GGGACCAGGTGCTGGCACCAGCAAGAGAGTGAACTGGACCGGTTACCAT

[0102] GTCATCAAAAGTGCAACAGAGGCAAGCAAGTTTACGGTGGCACAACTCA

[0103] TCCAAGGTGATGTTTGGTTGAAGAACACCGGGGTCAACTACATTGAGGG

[0104] ACTCTAG

[0105] SEQ ID NO.2

[0106] ATCAGATTTGCATGAATTTCTACTGGTACACCTTATGTTTCTAAACCGAAT

[0107] CAATCTGCCATCCACTCTGTTCTTTTGTGTCAAAAGAGACTCAACAAGCA

[0108] TGGGAAGAAAGAATGCTATTCTCTTTGGAGTTCTTCTTCAAAAGGTATTG

[0109] AACAATCAAATCCTTGATGCTATCACTGAACAACTATCAACCCTTCAAGG

[0110] ATTCTTTCAACCATGTCTGGAGAGACTCAATTTCGTGAGAATGAAACTAA

[0111] CACCAATTGAGAATGCTCAAAGAAAAAACTCAAATGGTGCTCAAGAGGC

[0112] CTCTGAAGAAAGGAGAAAATGAACAAAACTCTCGAACTGCAAGAGCG

[0113] GAGGACCAACGTGAAGCCCAAATCAGTAGAGCAAGAGAAGAGATCAGA

[0114] AAATTCAAGGTAGATCAGCTGGCTGCTGAAAGAAGAGAAAAAAAAGC

[0115] CAAAAAGAATAAGAGAAGAAGAAAAGGAAGTTGCTAAGCATATCCTGAAT

[0116] CTCTTGGATAAAATAAACAGAAAAAGGAATGCTGGGGACATGCTCACTC

[0117] CATCTTAAGCTGAACCATCCGCGATGAATGATGATTCCCTTGAAGTGTCC

[0118] ACATGTCAGATAAATATGAACACTTCTACTCGAGATATTGGTGATCAATC

[0119] TCTGACAGGAGCAGTAAGAAGGCCTTTTACCAGGTCTATGAGACAAGAG

[0120] CAAGATCAGTTGCCTGAAACTTCTTCACGGGAATGAAGATCCAAAAAAGA

[0121] GAGCTGATCGACTCTCTCAAATCTATCAAGAACCTGAGGCTGTAGCCGAC

[0122] AACACTCCTGTACCGGAAGCTGCTGCCGTAGCATCTGAGGAAGGAAATG

[0123] GTTTCCAAGGTCCTGTATCCCAGAGAAGTTTCTACGGCTATCGGCCAAA

[0124] GTGGACAGTCTGATAGCTCTGTTTCAAGATCTTTGTCAAGAAGAAGGATTA

[0125] AGAGATTTTCTATCTTTTCATATTCCTTGTTTTTTGTTTTTTTTTTTCTTCTCT

[0126] TCTTTTCTTCTTTTGTGGACTATGATGTTCTATGAACCTTTTTGTCTGATGA

[0127] AAAAGGGGGAAATTGGGAAATTAGGTTTTAAAGATCACAAGTCA

[0128] TTAATAAAATGATTGCTCTGTGAGAAAGAGAAATTTTTCAGGTACTCTGA

[0129] AC

[0130] SEQ ID NO.3

[0131] ATGGCTTCCAATAAGCTCAATGCCACCATCTTATTGCTTTCTCTCCTTGCC

[0132] TATTCATTAACATTCTCCCATGCCTGTTGCTCATGTAAGTCAAAGCCCAAA

[0133] ACACCTCCTAAGGCTCCTTGTACCACTCCTCCTACTACTCCTACTCCTACT

[0134] CCTACTACTCCAGCAGCACCAGCACCAGCATCAGGAAAGTGCCCCACTG

[0135] ACACACTGAAGCTAGGAGTTTGTGCTGACATTCTAGGACTTGTTAATGTT

[0136] GTTGTTGGATCCCCTGCTTCAAGCCAGTGCTGTGCATTGCTTGAAGGTTTG

[0137] GCTGATTTAGATGCAGCACTTTGTCTTTGCACTGCTATTAAGGCCAATGTG

[0138] CTTGGGATCAACCTGAATGTGCCTGTCACACTCAGCCTTCTACTTAGTGCT

[0139] TGTCAAAAAGATGTTCCTTCTGGTTTCAAATGTGCATAG

[0140] SEQ ID NO.4

[0141] ATGGCTTCCAAACGGATCTTGAAGGAACTCAAGGATCTTCAGAAGGATCC

[0142] TCCTACCTCATGCAGCGCCGGTCCTGTTCATGAGGACATGTTTCATTGGC

[0143] AAGCAACTATTATGGGTCCTCCAGACAGTCCTTATGCTGGGGGTGTTTTC

[0144] CTAGTCACAATTCATTTCCCTCCAGATTATCCCTTCAAGCCACCCAAGGTT

[0145] GCATTCAGGACGAAGGTATTTCACCCAAATATAAATAGCAATGGAAGCA

[0146] TTTGCCTTGACATATTGAAGGAGCAGTGGAGTCCTGCACTAACCATTTCC

[0147] AAGGTGTTGCTCTCAATTTGTTCCCTGTTGACGGACCCAAATCCCGATGA

[0148] TCCTTTGGTCCCCGAAATCGCCCACATGTACAAGACAGACAGGAACAAGT

[0149] ACGAGTCAACTGCCAGAAGTTGGACTCAGAAATATGCCATGGGTTAA

[0150] SEQ ID NO.5

[0151] ATGGCCTCAAGAGTAGCGTTGTTGTTGGTTATGTGTGTGCTTCCGGCGAT

[0152] GGTTGCAGCCATTCGCCCAGCGAAGAACCCTTTCTGCGTGAAGGGTCGCG

[0153] TCTACTGTGACCCCTGCCGTGCTGGTTTTGAGACCCCAGCCACCACCTAC

[0154] GTTGCTGGTGCGGAGGTTCTGCTGCAATGCAAGGACAAGAATAGCAACG

[0155] AAGTAGTGTACACAAAGCGGGGTTATACTGACTCAACAGGAGCATATAC

[0156] AATGTACGTGGATGAGGATCACGCAGACCAGATCTGTGATGCCAAGCTT

[0157] GTGAGCAGCCCTCAGCATGATTGCAAAGAAGCCACCCCAGGCCGCGACC

[0158] AAGCGCGCGTTATCCTCACTCGCTACAATGGCATCTCCTCCGACGACAGG

[0159] TTCGCCAATGCCATGGGATTCATGACTCAGGAGGTTGCCTCTGGCTGTGC

[0160] TGAGATCTTCAGGCAATACCAGGAGTTCGATAACGAGAACTAA

[0161] SEQ ID NO.6

[0162] GGATCCGTCGACATGGCTGGTAGAATGGATTTTTATAATTCTCCACAATT

[0163] GCATTCTGATCCATTTAGAGGTGGAGGTGAATTGATGGAAGTTTTGGAAC

[0164] CATTTATGAAATCTGCTTCTACTACAACTCCATCTTCATCTAACTCTCATT

[0165] TGCCATCTACTTCTACTTCTTTTTTTTATCCATCTCCAAATTCTTATCCAAA

[0166] ACCATCTTTGTCTTTGCCACCATTGCCAAATTTTTATACTACTGATAATGG

[0167] TTCTTATTTGTTTCCATCTTCTGAAACTTCTCAAAATTTGATTGGTTTTGAA

[0168] CAACCATCTTCAGTTTTGGGATTAAATAACTTAACTCCATATCAAATTAA

[0169] CCAAATCCAATCTCAAATTCAATTGCAAGCTCAAGTTCAAGCTCATCATA

[0170] ATATTAATACTTTGTCTTTTTTGGGTCCAAAACCAATTTCTATGAAACAAG

[0171] TTGGTGTTCCACCTCCACCAAAACCAACTAAATTGTATAGAGGTGTTAGA

[0172] CAAAGACATTGGGGTAAATGGGTTGCTGAAATTAGATTGCCAAAAAATA

[0173] GAACTAGATTGTGGTTGGGTACTTTTGATACTGCTGAAGAAGCTGCTTTG

[0174] GCTTATGATAGAGCTGCTTATAGATTGAGAGGTGATTTTGCTAGATTGAA

[0175] TTTTCCAAATTTGAAGAATCATGGTTCTTTTGGTGAATCTTTTAAACCATT

[0176] GCATTCTTCTATTGATGCTAAATTGGATGCTATTTGTGAGAACTTGGCAG

[0177] ATATGCAAAAACAAGAAAAAATTGAAAAAAAGGGAGCTAGATCTTCTTC

[0178] AAAAAAAACTGGTTCTAAAGAAGTTCAACCACAAGCTTCTTTGTCTCCTG

[0179] TTATGACTGAATCTGATGAAGGTTCTGCTGATTCTTCTCCATTGTCTGATT

[0180] TGACTTTTGGTGATGTTACTGAACCACAATGGGAAGCTGGTTCTGAACCA

[0181] TTTAATTTGCAAAAATTTCCATCTTATGAAATTGATTGGGATTCTTTGTAA

[0182] SEQ ID NO.7

[0183] ATGGCTCCAAGGGACAGAGCCACCGCACTCGCTACAGGGCCCATTAACC

[0184] CAACCACCACAACCGTCAAGGAGATCCGTTACAGGGGCGTTAGGAAGCG

[0185] CCCTTGGGGCCGTTACGCCGCCGAGATCCGCGACCCCGGGAAGAAAACA

[0186] CGTGTCTGGCTCGGCACCTTCGACACCGCCGAGGAGGCCGCGCGTGCCTA

[0187] TGACGCGGCCGCTAGAGAGTTTCGCGGCGCGAAGGCAAAGACGAATTTC

[0188] CCGACGCCTTCGGAGCTTATAATGAACAACTCCGCCCGCAGCCCCAGCCA

[0189] GAGCAGCACCCTTGAGTCACCCTCTCCGCCGCCGCTGGACCTGACCCTCA

[0190] CACCTCTCTCCGTCGCCGGAGGCGGTTGTTCCGGCGGCGCCGCCATGGGT

[0191] TTTCATGTTGCGCGCCCTGTTTTGTTCTTCGACGCTTTTGCGCGCGCTGAC

[0192] ACCGGGCTGAGCGTTGGTCGTCGCGAGATGTGCGGGTTCGAACTTCCGGT

[0193] GGCTGATTTTCGACGCGCCGCCGCCCAGAGCGACTCCGATTCTTCCTCCG

[0194] TCGTGGACTGCGAGCGTGTCCCGCGTCAGAGACTCTTGGATCTCGACCTC

[0195] AACGTCCCTCCTCCTCCGGAGGTTGCTTGA

[0196] SEQ ID NO.8

[0197] ATGGCGCCGAGAGATAAGACGGTTGGCGTTAAGGTGAACGGCAACGGTA

[0198] GCGGCAACGGCGGCGTGAAGGAGGTGCACTTTCGGGGAGTGAGGAAGAG

[0199] GCCGTGGGGGAGATACGCCGCGGAGATCAGAGATCCCGGCAAGAAGAGC

[0200] CGCGTCTGGCTCGGAACCTTCGACACGGCGGAGGAGGCGGCGCGAGCCT

[0201] ACGACACCGCCGCACGCGAATTCCGCGGCCCCAAGGCCAAAACCAACTT

[0202] CCCCCTCCCTTCGGACAATGTCAAGATCCAGAGCCCCAGCCAGAGCAGCA

[0203] CCGTCGAATCCTCCAGCCGCGACCGTGACGCCGCCGCTGACTCCTCCCCG

[0204] CTCGATCTCAACTTGGCCCCCGCCGTCTCCGTCGCCGGCCCCGTTCGGTTC

[0205] CCCTTCCAGCACCAGTTCCCGCCGGTTCCGGCGGCTAACCAGGTGTTTTA

[0206] CTTCGACGCCGTCCTACGCGCCGGCATGGTGGGTCCCCGTCCCTGCCAGG

[0207] GATTCGGGTTCAATTACAATCATCCCGTGGCGGCGAGTGAGTTCCACGCG

[0208] ACTGCAGGAGCACAGAGCGACTCGGACTCGTCCTCCGTGATCGATCTGAA

[0209] CCACCACCATGATGGCGATGTTAAGGCCGTTAGAGACTTCGATCTCGACC

[0210] TTAACTACCCTCCTCCGGTGGAGATGCCTTGA

[0211] SEQ ID NO.9

[0212] ATGAAGAGGGGAAGAGGAGGTGCTGCGGCGGCGGCGGAGGAAGTGAAC

[0213] GGATCGGCGGTAGCAATAAAAGAGCCACGTTACAGGGGAGTGAGGAAG

[0214] AGACCGTGGGGGAGATTTGCGGCGGAGATAAGGGATCCATTGAAGAAAG

[0215] CGAGGGTGTGGCTTGGAACATTCGATTCCGCCGAGGAAGCTGCTCGAGCC

[0216] TACGACGCAGCCGCACGAACACTCCGTGGACCTAAGGCTAAGACCAATT

[0217] TCCCTCTCTCCCCTTTCTGTTACCACCCTCACAATACCAACAACAATAACA

[0218] ACTCCGATCCGTTTCTCTACGGTTTCCAACAACAACAGCCTCAGAGACCT

[0219] ACCTCCAGTGGCATGAGTAGCACCGTTGAGTCCTTCAGTGGGCCCCGCCC

[0220] TCCCTCAACTAAACCTCAAACGCCGTCGTTTTTGCCAACACCCGCTCGGC

[0221] GCTACCCTCGCACGCCGCCACTCGTCCCTGAAGACTGCCGCAGCGACTGC

[0222] GACTCCTCTTCCTCCGTCGTTGACGATGACGGCGATAACAACATCGTTAA

[0223] CTCGTCGTTTAGACCCCCTTTGCCTTTCGATCTAAACGCACTGCCGTTCGA

[0224] TGATGCTGACGTGGCTGCTGCTTCTGCCGGTGACGACAACTCTGATCTTC

[0225] GCTGCACTGCGCTCTGCCTTTGA

Claims

1. A licorice-derived gene element EC11 Its application in enhancing the tolerance of brewer's yeast to high temperature and acetic acid stress is characterized by: The licorice-derived gene element EC11 The nucleotide sequence is SEQ ID NO.1; The high temperature is 37°C; the amount of acetic acid added is 0.3%.

2. A kind EC11 The expression box is characterized by: Contains the licorice-derived gene element as described in claim 1 EC11 .

3. As described in claim 2 EC11 The expression box is characterized by: The EC11 The promoter in the expression box is TDH3 The promoter and terminator are CYC1 And it was labeled with ampicillin antibiotic.

4. As described in claim 2 EC11 The expression box is characterized by: The EC11 The integration site of the expression cassette with the yeast genome is HO .

5. A receptor cell, characterized in that: Contains the contents of claim 2 EC11 Expression box.

6. The receptor cell as described in claim 5, characterized in that: Will EC11 The expression cassette was introduced into recipient cells via electroporation, and the recipient cells were Saccharomyces cerevisiae BY4742.

7. A method for improving the tolerance of brewer's yeast to high temperature and acetic acid stress, characterized in that: The licorice-derived gene element as described in claim 1 EC11 The yeast was overexpressed in Saccharomyces cerevisiae BY4742 at a high temperature of 37°C and the amount of acetic acid added was 0.3%.

8. The licorice-derived gene element as described in claim 1 EC11 Application in the production of bioethanol by fermentation of Saccharomyces cerevisiae.

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