Application of yeast initiation transcription factor mutant protein and derivative product thereof in production of industrial ethanol

Abstract

The invention provides application of yeast initiation transcription factor mutant protein and derivative products thereof in production of industrial ethanol, and belongs to the technical field of microbial gene modification. A yeast transcription factor (spt15) is mutated by using a gTME technology, a gene mutation library is encoded, a yeast spt15 mutant protein of which the 127th lysine of a yeast initial transcription factor is mutated into glycine, glutamine, serine, isoleucine and aspartic acid is obtained, and a recombinant saccharomyces cerevisiae strain is constructed by using a genetic engineering means. Through fermentation environment parameter tolerance detection, the saccharomyces cerevisiae strain with at least two of acetic acid resistance, ethanol resistance, high temperature resistance and high permeability resistance is obtained, and a new means is provided for efficient fermentation production of industrial ethanol.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial gene modification, and in particular relates to application of a yeast initiation transcription factor mutant protein and a derivative product thereof in the production of industrial ethanol. Background Art

[0002] During the bioethanol fermentation process, high stress environments such as high ethanol concentration, high sugar, high temperature, low pH and toxic byproducts often occur. Severe inhibition will lead to premature termination of fermentation. Therefore, obtaining yeast strains with improved tolerance to high stress environments can improve production efficiency and save costs. This is especially important in ultra-high concentration ethanol fermentation processes and cellulosic ethanol production processes, and is a core task in modern ethanol industrial production. At present, the selection and breeding of brewer's yeast strains mainly include natural selection, adaptive domestication, mutation breeding and genetic engineering. In the late fermentation of yeast, ethanol becomes the main stress factor. With the research of genome, transcriptome and other multi-omics, it is shown that ethanol tolerance is subject to complex regulation of multiple genes and networking, and the number of genes that may change in expression levels may reach hundreds or thousands. The complexity of the molecular mechanism of ethanol tolerance means that it is difficult to obtain a high ethanol tolerance phenotype by modifying a single gene or metabolic pathway. Therefore, the technical means of modifying the overall metabolic network of cells are particularly important.

[0003] The method of transforming global transcription regulatory factors using global transcription machinery engineering (gTME) technology, using directed evolution technology to randomly mutate and direct the transcription factors, and achieve fine regulation of gene transcription levels, can achieve moderate regulation of metabolic networks, achieve balanced evolution of cell metabolic networks, and improve alcohol production or stress tolerance at the overall level of cells. This is the research direction of yeast metabolic engineering. Transcription factor spt15 is a TATA binding protein. As the first complex protein bound to DNA, TATA binding protein plays a very important role in the assembly and transcription of the pre-initiation complex. After binding to RNA polymerase II and more than a dozen basic transcription factors, it can directly transcribe the mRNA of most related genes in the genome. However, the gene results of the mutant protein obtained by site-directed mutation of transcription factor spt15 are not clear. At present, there are very limited reports on mutant proteins resistant to high concentration ethanol, high temperature, high osmotic pressure, and high acetic acid content obtained by mutating transcription factor spt15, which undoubtedly hinders the production of industrial ethanol. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a yeast initiation transcription factor mutant protein, a coding gene of the yeast initiation transcription factor mutant protein or a genetic modification product comprising the coding gene for use in the industrial production of ethanol. By performing point mutation modification on the initiation transcription factor coding gene spt15, the tolerance of the yeast strain to acetic acid concentration, high permeability, high temperature and ethanol concentration is improved, thereby amplifying the method for industrial production of ethanol.

[0005] The present invention provides a yeast initiation transcription factor mutant protein, a coding gene of the yeast initiation transcription factor mutant protein or a genetic modification product containing the coding gene in the industrial production of ethanol;

[0006] The yeast initiation transcription factor mutant protein is obtained by mutation of the 127th lysine of the yeast initiation transcription factor spt15 to one of the following amino acid residues: glycine, glutamine, serine, isoleucine and aspartic acid.

[0007] Preferably, the amino acid sequence of the yeast initiation transcription factor spt15 is as shown in SEQ ID NO: 1;

[0008] Preferably, when mutated to glycine, the yeast initiation transcription factor mutant protein is spt15-G, and the amino acid sequence is shown in SEQ ID NO: 2;

[0009] When mutated to glutamine, the yeast initiation transcription factor mutant protein is spt15-Q, and the amino acid sequence is shown in SEQ ID NO: 3;

[0010] When mutated to serine, the yeast initiation transcription factor mutant protein is spt15-S, and the amino acid sequence is shown in SEQ ID NO: 4;

[0011] When mutated to isoleucine, the yeast initiation transcription factor mutant protein is spt15-I, and the amino acid sequence is shown in SEQ ID NO:5;

[0012] When mutated to aspartic acid, the yeast initiation transcription factor mutant protein is spt15-D, and the amino acid sequence is shown in SEQ ID NO:6.

[0013] Preferably, the nucleotide sequences of the genes encoding the yeast initiation transcription factor mutant proteins spt15-G, spt15-Q, spt15-S, spt15-I and spt15-D are shown in at least one of SEQ ID NO: 16 to SEQ ID NO: 20.

[0014] Preferably, the genetically modified product comprising the encoding gene comprises a recombinant vector comprising the encoding gene, a recombinant yeast strain comprising the encoding gene, or a recombinant yeast strain comprising the recombinant vector.

[0015] Preferably, when the yeast initiation transcription factor mutant protein is spt15-G or spt15-Q, during the process of producing industrial ethanol by the recombinant yeast strain, the acetic acid concentration in the fermentation system is 5-6 g / L.

[0016] Preferably, when the yeast initiation transcription factor mutant protein is spt15-G or spt15-S, during the process of producing industrial ethanol by the recombinant yeast strain, the mass percentage of sodium chloride in the fermentation system is 7% to 9%.

[0017] Preferably, when the yeast initiation transcription factor mutant protein is spt15-I or spt15-S, during the production of industrial ethanol by the recombinant yeast strain, the fermentation temperature is 41-43°C.

[0018] Preferably, when the yeast initiation transcription factor mutant protein is spt15-G, spt15-S or spt15-D, during the process of producing industrial ethanol by the recombinant yeast strain, the volume percentage of ethanol in the fermentation system is 14% to 19%.

[0019] The present invention provides a yeast initiation transcription factor mutant protein, a coding gene of the yeast initiation transcription factor mutant protein or a recombinant vector containing the coding gene for use in improving yeast tolerance to a fermentation environment. The yeast initiation transcription factor mutant protein is the yeast initiation transcription factor mutant protein in the application.

[0020] Preferably, when the yeast initiation transcription factor mutant protein is spt15-G or spt15-Q, the yeast initiation transcription factor mutant protein, the encoding gene of the yeast initiation transcription factor mutant protein or the recombinant vector is used to improve the tolerance of yeast to acetic acid;

[0021] When the yeast initiation transcription factor mutant protein is spt15-G or spt15-S, use of the yeast initiation transcription factor mutant protein, the encoding gene of the yeast initiation transcription factor mutant protein or the recombinant vector in improving yeast tolerance to sodium chloride;

[0022] When the yeast initiation transcription factor mutant protein is spt15-I or spt15-S, use of the yeast initiation transcription factor mutant protein, the encoding gene of the yeast initiation transcription factor mutant protein or the recombinant vector in improving yeast tolerance to fermentation temperature;

[0023] When the yeast initiation transcription factor mutant protein is spt15-G, spt15-S or spt15-D, the yeast initiation transcription factor mutant protein, the encoding gene of the yeast initiation transcription factor mutant protein or the recombinant vector are used in improving yeast tolerance to ethanol.

[0024] The present invention provides a yeast initiation transcription factor mutant protein, a coding gene of the yeast initiation transcription factor mutant protein or a genetic modification product containing the coding gene in the industrial production of ethanol; the yeast initiation transcription factor mutant protein is a yeast initiation transcription factor spt15 in which the 127th lysine is mutated to one of the following amino acid residues: glycine, glutamine, serine, isoleucine and aspartic acid. The present invention performs single-point mutation on the yeast spt15 gene to screen mutant genes with multiple tolerances. The experiment showed that the yeast spt15 mutant gene was transformed into yeast through a recombinant vector to express a mutant protein of the initiation transcription factor. After verification by tolerance experiments, the results showed that INVSC1-spt15-G and INVSC1-spt15-Q had good tolerance to 6 g / L acetic acid, and their tolerances were 3.64 times and 4.79 times that of the INVSC1-Neo control strain, respectively; INVSC1-spt15-G and INVSC1-spt15-S had good tolerance to the high osmotic pressure of sodium chloride with a mass fraction of 9%, and their tolerances were 1.16 times and 1.75 times that of the INVSC1-Neo control strain, respectively; the recombinant strain INVSC1-spt15-I had good tolerance to a high temperature of 42°C, and its tolerances were 1.58 times and 1.45 times that of the control strain INVSC1-Neo, respectively. INVSC1-spt15-G, INVSC1-spt15-S, and INVSC1-spt15-D have good tolerance to 16% ethanol solution, and their tolerance is 2.76 times, 4.17 times, and 3.08 times that of the INVSC1-Neo control group, respectively. It can be seen that the yeast spt15 mutant gene of the present invention can improve the yeast's good tolerance to at least one of acetic acid, high osmotic pressure, high temperature, and ethanol, providing a basis for genetic engineering to construct tolerant yeast. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The three-dimensional structure of spt15 and the schematic diagram of the mutation site structure;

[0026] Figure 2The results of cloning the spt15 gene and verifying the pYES2NTc-spt15 recombinant plasmid are shown in Figure A. 1: PCR product of spt15; M: DNAmarker DL2000; Figure B: 1: double enzyme digestion verification of pMD18-spt15 plasmid; M: DNAmarker DL10000; Figure C: 1: PCR verification of pYES2NTc-spt15 plasmid; 2: pYES2NTc-spt15 plasmid; 3: single enzyme digestion product of pYES2NTc-spt15; M: DNAmarker DL5000;

[0027] Figure 3 The growth results of the recombinant strain on the YPD plate containing different concentrations of acetic acid; A is the growth result of the recombinant strain on the YPD plate containing 5 g / L acetic acid, and B is the growth result of the recombinant strain on the YPD plate containing 6 g / L acetic acid;

[0028] Figure 4 is the growth curve of the recombinant strain in medium containing acetic acid;

[0029] Figure 5 The growth results of the recombinant strain on the YPD plate containing NaCl; A is a plate with a NaCl content of 7%, and B is a plate with a NaCl content of 9%;

[0030] Figure 6 is the growth curve of the recombinant strain in the medium containing NaCl;

[0031] Figure 7 The growth results of the recombinant strain on YPD plates at different temperatures; A is a plate cultured at 41°C, and B is a plate cultured at 43°C;

[0032] Figure 8 is the growth curve of the recombinant strain in culture medium at different temperatures;

[0033] Fig. 9 The growth results of the recombinant strain on the YPD plate containing ethanol; A is a plate with an ethanol volume fraction of 14%, and B and C are plates with an ethanol volume fraction of 16%;

[0034] Fig.10 This is the growth curve of the recombinant strain in ethanol-containing medium. DETAILED DESCRIPTION

[0035] The present invention provides a yeast initiation transcription factor mutant protein, a coding gene of the yeast initiation transcription factor mutant protein or a genetic modification product containing the coding gene, and use thereof in industrial production of ethanol; the yeast initiation transcription factor mutant protein is a yeast initiation transcription factor spt15 in which the 127th lysine is mutated to one of the following amino acid residues: glycine, glutamine, serine, isoleucine and aspartic acid.

[0036] In the present invention, the amino acid sequence of the yeast initiation transcription factor spt15 is preferably as shown in SEQ ID NO: 1

[0037]

[0038] When mutated to glycine, the yeast spt15 mutant gene is preferably spt15-G, and the amino acid sequence is shown in SEQ ID NO: 2 (MADEERLKEFKEANKIVFDPNTRQVWENQNRDGTKPATTFQSEEDIKRAAPESEKDTSATSGIVPTLQNIVATVTLGCRLDLKTVALHARNAEYNPKRFAAVIMRIREPKTTALIFASGKMVVTGAGSEDDSKLASRKYARIIQKIGFAAKFTDFKIQNIVGSCDVKFPIRLEGLAFSHGTFSSYEPELFPGLIYRMVKPKIVLLIFVSGKIVLTGAKQREEIYQAFEAIYPVLSEFRKM). When mutated to glutamine, the yeast spt15 mutant gene is preferably spt15-Q, and the amino acid sequence is shown in SEQ ID NO: 3

[0039]

[0040] When mutated to serine, the yeast spt15 mutant gene is preferably spt15-S, and the amino acid sequence is as shown in SEQ ID NO: 4

[0041]

[0042] When mutated to isoleucine, the yeast spt15 mutant gene is preferably spt15-I, and the amino acid sequence is as shown in SEQ ID NO: 5

[0043]

[0044] when mutated to aspartic acid, the yeast spt15 mutant gene is preferably spt15-D, and the amino acid sequence is shown in SEQ ID NO:6 (MADEERLKEFKEANKIVFDPNTRQVWENQNRDGTKPATTFQSEEDIKRAAPESEKDTSATSGIVPTLQNIVATVTLGCRLDLKTVALHARNAEYNPKRFAAVIMRIREPKTTALIFASGKMVVTGADSEDDSKLASRKYARIIQKIGFAAKFTDFKIQNIVGSCDVKFPIRLEGLAFSHGTFSSYEPELFPGLIYRMVKPKIVLLIFVSGKIVLTGAKQREEIYQAFEAIYPVLSEFRKM).

[0045] In the present invention, the yeast initiation transcription factor mutant protein encoding gene is a yeast spt15 mutant gene, which is a yeast spt15 gene with mutations at bases 379 to 381. The nucleotide sequence of the yeast spt15 gene is shown in SEQ ID NO: 7

[0046] When the mutation is to a codon encoding glycine, the codon mutates to GGG, and the corresponding nucleotide sequence of the coding gene spt15-G is shown in SEQ ID NO: 16

[0047] When the mutation is to a codon encoding glutamine, the codon is mutated to CAG, and the corresponding nucleotide sequence of the coding gene spt15-Q is shown in SEQ ID NO: 17

[0048] When the mutation is to a codon encoding serine, the codon is mutated to TCA, and the corresponding nucleotide sequence of the coding gene spt15-S is shown in SEQ ID NO: 18

[0049] When the mutation is to a codon encoding isoleucine, the codon is mutated to ATT, and the corresponding nucleotide sequence of the coding gene spt15-I is shown in SEQ ID NO: 19.

[0050] When the mutation is to the codon encoding aspartic acid, the codon is mutated to GAT, and the corresponding nucleotide sequence of the coding gene spt15-D is shown in SEQ ID NO: 20.

[0051] (ATGGCCGATGAGGAACGTTTAAAGGAGTTTAAAGAGGCAAACAAGATAGTGT ).

[0052] In the present invention, the mutation method of the yeast spt15 mutant gene preferably uses yeast genomic DNA as a template to amplify the yeast spt15 gene; the yeast spt15 gene is subjected to mutation amplification using a saturation site mutation primer pair to obtain a PCR product; the PCR product is detected to screen for a mutant gene encoding an amino acid residue of glycine, glutamine, serine, isoleucine and aspartic acid.

[0053] In the present invention, the primers for amplifying the yeast spt15 gene are preferably a forward primer having a nucleotide sequence as shown in SEQ ID NO:8 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:9. After obtaining the yeast spt15 gene, the amplified yeast spt15 gene is preferably cloned into a vector to prepare a recombinant vector, and the recombinant vector is transformed into Escherichia coli for propagation, and the recombinant vector is extracted. The vector is preferably pMD18-T. After obtaining the extracted recombinant vector, the yeast spt15 gene is preferably cloned into a yeast expression vector to obtain a recombinant expression yeast vector. The yeast expression vector is preferably pYES2NTc. When the yeast spt15 gene is cloned into the yeast expression vector, the primers for amplifying the yeast spt15 gene are preferably Spt15_yes2nt (SEQ ID NO:10) and Spt15_yes2ntR (SEQ ID NO:11). The saturation site mutation primer pair is preferably Spt15_mutF (SEQ ID NO:12) and Spt15_mutR (SEQ ID NO:13). The reaction conditions of the mutation amplification are preferably: 94°C 50 sec, 62°C 45 sec, 72°C 7 min (30 cycles); 72°C extension for 10 min. The screening method is preferably sequencing screening, and the recombinant vector containing the target mutant gene is selected according to the sequencing results.

[0054] In the present invention, the genetic modification product containing the coding gene preferably includes a recombinant vector containing the coding gene, a recombinant yeast strain containing the coding gene, or a recombinant yeast strain containing the recombinant vector. The recombinant vector containing the coding gene is a recombinant vector containing the yeast spt15 mutant gene. The backbone vector of the recombinant vector is preferably a prokaryotic expression vector and / or a eukaryotic expression vector. The prokaryotic expression vector preferably includes pMD18-T, which is used for rapid amplification of the yeast spt15 mutant gene. The eukaryotic expression vector preferably includes pYES2NTc, which is used to mediate the yeast spt15 mutant gene into yeast cells for expression. The construction method of the recombinant vector is the same as described above and will not be repeated here. The recombinant yeast strain containing the coding gene is preferably a recombinant yeast strain containing the yeast spt15 mutant gene or the recombinant vector. The host cell of the recombinant yeast strain preferably includes Saccharomyces cerevisiae. The strain of Saccharomyces cerevisiae is INVSC1.

[0055] In the present invention, when the yeast spt15 mutant gene is preferably spt15-G and / or spt15-Q, the concentration of acetic acid in the fermentation system during the industrial production of ethanol by the recombinant yeast strain is preferably 5 to 6 g / L, more preferably 5.5 to 6 g / L. In one embodiment of the present invention, INVSC1-spt15-G and INVSC1-spt15-Q can tolerate acetic acid at a concentration of 6 g / L and grow well under the condition of an acetic acid concentration of 6 g / L, while the INVSC1-Neo control strain cannot grow under the condition of an acetic acid concentration of 6 g / L, and the OD value is substantially unchanged. Under the condition of a medium with an acetic acid concentration of 6 g / L, the acetic acid tolerance of INVSC1-spt15-G and INVSC1-spt15-Q is 3.64 times and 4.79 times that of INVSC1-Neo when cultured for 48 hours, respectively, wherein the tolerance of INVSC1-spt15-Q to acetic acid is slightly higher than that of INVSC1-spt15-G.

[0056] In the present invention, when the yeast spt15 mutant gene is preferably spt15-G and / or spt15-S, the concentration of sodium chloride in the fermentation system during the industrial production of ethanol by the recombinant yeast strain is preferably 8% to 9%, and most preferably 8.5% to 9%. In one embodiment of the present invention, the control strain INVSC1-Neo cannot grow under conditions greater than 7%, and the mutant recombinant strains can grow in a culture medium with a NaCl mass fraction of 7%. When inoculated into a culture medium with a NaCl mass fraction of 9%, only INVSC1-spt15-G and INVSC1-spt15-S strains can grow. Under the condition of a culture medium with a NaCl mass fraction of 9%, when cultured for 48 hours, the tolerance of the recombinant strains INVSC1-spt15-G and INVSC1-spt15-S to NaCl is 1.16 times and 1.75 times that of the INVSC1-Neo control strain, respectively.

[0057] In the present invention, when the yeast spt15 mutant gene is preferably spt15-I and / or spt15-S, the fermentation temperature during the industrial production of ethanol by the recombinant yeast strain is 41°C to 43°C. In one embodiment of the present invention, the high temperature resistance condition of the control bacterium INVSC1-Neo is 41°C. Taking the culture temperature of 41°C as the standard, the culture temperature is increased successively. Only the two strains INVSC1-spt15-I and INVSC1-spt15-M can grow under the condition of a culture temperature of 43°C, while other strains cannot grow under this condition. Under the condition that the fermentation medium temperature is 42°C, the tolerance of INVSC1-spt15-I is 1.58 times that of the control strain INVSC1-Neo.

[0058] In the present invention, when the yeast spt15 mutant gene is preferably spt15-G, spt15-S and / or spt15-D, the concentration of ethanol in the fermentation system during the industrial production of ethanol by the recombinant yeast strain is preferably 14% to 16%, more preferably 15% to 16%. In one embodiment of the present invention, the control bacteria can grow in a culture medium with an ethanol volume fraction of 14%. When inoculated into a culture medium with an ethanol volume fraction of 16%, only the recombinant strains INVSC1-spt15-G, INVSC1-spt15-S and INVSC1-spt15-D strains can grow, indicating that only these three strains have acquired a certain ethanol tolerance. Under the condition of adding 16% ethanol by volume to the culture medium, when cultured for 48 hours, the ethanol tolerance of INVSC1-spt15-G, INVSC1-spt15-S and INVSC1-spt15-D is 2.76 times, 4.17 times and 3.08 times that of the INVSC1-Neo control group, respectively. The strains were arranged from high to low in terms of tolerance as follows: INVSC1-spt15-S, INVSC1-spt15-D, and INVSC1-spt15-G.

[0059] The invention provides a yeast initiation transcription factor mutant protein, a coding gene of the yeast initiation transcription factor mutant protein or a recombinant vector containing the coding gene for improving the tolerance of yeast to a fermentation environment.

[0060] In the present invention, when the yeast spt15 mutant gene is spt15-G and / or spt15-Q, the yeast spt15 mutant gene or the recombinant vector is preferably used to improve the tolerance of yeast to acetic acid; the tolerance concentration of acetic acid is 5 to 6 g / L, more preferably 5.5 to 6 g / L.

[0061] In the present invention, when the yeast spt15 mutant gene is spt15-G and / or spt15-S, the yeast spt15 mutant gene or the recombinant vector is preferably used to improve the yeast's tolerance to sodium chloride; the tolerance mass concentration of sodium chloride is 8% to 9%, and most preferably 8.5% to 9%.

[0062] In the present invention, when the yeast spt15 mutant gene is spt15-I and / or spt15-S, the yeast spt15 mutant gene or the recombinant vector is preferably used to improve the tolerance of yeast to fermentation temperature; the fermentation temperature is preferably 41-43°C, more preferably 42-43°C.

[0063] In the present invention, when the yeast spt15 mutant gene is spt15-G, spt15-S and / or spt15-D, the yeast spt15 mutant gene or the recombinant vector is preferably used to improve the tolerance of yeast to ethanol. The tolerance volume concentration of ethanol is 14% to 16%, more preferably 15% to 16%.

[0064] The application of a yeast initiation transcription factor mutant protein and its derivatives provided by the present invention in the production of industrial ethanol is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0065] Example 1

[0066] A mutation method for yeast spt15 mutant gene

[0067] 1. Cloning method of spt15 gene

[0068] The PCR amplification of the Saccharomyces cerevisiae initiation transcription factor spt15 gene was performed using Spt15-F / Spt15-R primers. The PCR cycle parameters were: 94℃5min; 94℃1min, 56℃1min, 72℃2min, 30 cycles.

[0069] The specific sequences of primers used for PCR amplification are as follows:

[0070] Spt15-F: 5'-ATGGCCGATGAGGAACGTTTAAAGGAGTTTA-3' (SEQ ID NO: 8);

[0071] Spt15-R5'-TCACATTTTTCTAAATTCACTTAGCACAGGGTATATAG-3' (SEQ ID NO: 9).

[0072] The spt15 gene amplified by PCR was separated by gel electrophoresis, and the separated product was placed in an agar block and placed in a 1.5 mL centrifuge tube. The target fragment was recovered using an ultra-thin agarose gel recovery kit, and the operation was performed according to the method in the instruction manual. The PCR product of the spt15 gene after recovery was connected to the pMD18-T vector, and the connection reaction system was placed at 16°C for connection overnight. The connection product was placed at 4°C for transformation, and the connection liquid was transformed into Escherichia coli E.coli DH5α competent cells by the calcium chloride method to extract the monoclonal plasmid. Finally, the E.coli DH5α cell transformation product was coated on an LB plate containing Amp100μg / ml and X-Gal40μg / ml, and the plasmid was extracted after inoculation in the culture medium.

[0073] 2. Construction of pYES2NTc-spt15 plasmid

[0074] After the correct spt15 gene sequence was obtained by sequencing, Spt15_yes2nt and Spt15_yes2ntR were used to PCR amplify the spt15 gene on the pMD18-spt15 plasmid.

[0075] Spt15_yes2ntF: 5'-GGATCCGCCGATGAGGAACGTTTA-3' (SEQ ID NO: 10);

[0076] Spt15_yes2ntR: 5'-GCGGCCGCTCACATTTTTCTAAATTCAC-3' (SEQ ID NO: 11). The PCR cycle parameters were: 94°C for 5 min; 94°C for 1 min, 56°C for 1 min, 72°C for 2 min, and 30 cycles.

[0077] Use gel to recover the spt15 gene and the pYES2NTc linear vector after double digestion with NotⅠ and BamHⅠ. The operation steps of the gel kit are as described in the instructions. Connect the spt15 gene fragment and the pYES2NTc linear vector. The 10μL connection reaction system is as follows: 1μL T4 DNA ligase solution, 1μL T4 DNA ligase Buffer solution, 4μL spt15 fragment, and 4μL pYES2NTc linear vector. Connect spt15 and pYES2NTc plasmid vectors and place them at 16℃ for connection overnight. The connection product is transformed at 4℃. The calcium chloride method is used for transformation. The connection solution is transformed into Escherichia coli DH5α competent cells, inverted in an incubator at 37℃, and cultured for 16h. The colonies that can grow are positive clones. Construction of recombinant Saccharomyces cerevisiae strains by global transcription factor directed evolution method

[0078] Pick the transformed E. coli DH5α colonies on the plate, inoculate the selected colonies into 4 ml of LB liquid medium, and then extract the plasmid. The obtained plasmid is double-digested with NotⅠ and BamHI to verify the acquisition of the site-directed saturation mutation gene library.

[0079] 3. Determine the mutation site

[0080] More than 50 spt15 core transcription factor genes from different yeast species were collected and sorted, and bioinformatics analysis of molecular evolution, protein three-dimensional structure and conserved regions was performed. Through research, it was found that the genes of the same species have small differences and high homology, and different species have different differences. Through homology comparison and three-dimensional structure analysis of spt15 sequences, such as Figure 1As shown, the results showed that the Lys127 site was located in the middle binding site of the symmetrical structure. Mutation of this site may affect the binding of spt15 to the target, so this study designed this site as a mutation site.

[0081] 4. Overlap Extension PCR

[0082] According to the principle of overlap extension PCR, a pair of mutation primers Spt15_mutF and Spt15_mutR were designed.

[0083] Spt15_mutF: 5'-ATGGTTGTTACCGGTGCAnnkAGTGAGGATGACTC A-3' (SEQ ID NO: 12);

[0084] Spt15 mutR: 5'-TGCACCGGTAACAACCATTTTCCCTGAGGCATTAA AGC-3' (SEQ ID NO: 13).

[0085] First, the recombinant plasmid pYES2NTc-spt15 was used as a template and primers Spt15_mutF and Spt15_mutR were used for plasmid amplification. The PCR reaction conditions were: 94°C for 50 sec, 62°C for 45 sec, and 72°C for 7 min (30 cycles). Finally, the linear fragment containing the vector sequence and gene sequence was obtained by reverse amplification.

[0086] 5. Obtaining a site-directed saturation mutagenesis library

[0087] After the template was digested by DpnⅠ enzyme, the PCR product was recovered by ultrathin agarose gel. The 10μL ligation reaction system was as follows: 1μL T4 DNA ligase solution, 1μL T4 DNA ligase Buffer solution, 7μL spt15 mutant fragment, 1μL pYES2NTc linear vector. E. coli DH5α was transformed, and after transformation, E. coli DH5α was coated with LB (containing Amp100μg / ml) medium. The colonies on the transformation plate were inoculated in 5ml (containing Amp100μg / ml) LB liquid medium, and then the plasmid was extracted. At the same time, it was sent to Invitrogen for sequence determination and sequencing to identify whether it was a mutant gene.

[0088] 6. Obtaining Recombinant Saccharomyces cerevisiae

[0089] The recombinant plasmids of the site-saturation mutagenesis gene library obtained after sequencing and pYES2NTc-spt15 were transformed into Saccharomyces cerevisiae INVSC1 by electroporation. The transformation method is as follows:

[0090] ① Activate the Saccharomyces cerevisiae strain INVSC1 to be transferred, then inoculate it into YPD medium and culture it at 30°C and 250 rpm for 16 hours.

[0091] ② The strain was inoculated again into 250 ml of fresh YPD medium and cultured at 30°C and 160 rpm for 16 h (OD 600 1.2~1.5).

[0092] ③ Place the cultured bacterial solution on ice for 15 minutes to ensure that it does not grow any more. Collect the supernatant and centrifuge at the highest speed for 5 minutes at 4°C. Pour the culture solution into a test tube and invert the tube for 1 minute to ensure that all the culture solution flows out.

[0093] ④ Resuspend the bacteria in the sterilized distilled water in the sterilizer and centrifuge at the maximum speed for about 1 minute.

[0094] ⑤Repeat the previous step, resuspend the bacteria again, and centrifuge at 4°C, 4000 rpm for 5 min.

[0095] ⑥ Resuspend the cells in 20 ml of ice-cold D-sorbitol (1 M) and centrifuge at 4°C and 4000 rpm for 5 min.

[0096] ⑦ Add 600 μL of ice-cold D-sorbitol (1 M) to resuspend the cells, mix thoroughly, and place on ice for use on the same day.

[0097] ⑧Pipette an appropriate amount of competent bacteria, mix thoroughly with 5 μL of plasmid, then place in an electroporation cup, and finally place on ice for 5 minutes.

[0098] ⑨Select yeast parameters and click Pulse conversion.

[0099] ⑩Immediately add 600μL ice-cold D-sorbitol (1M) prepared on the same day to the transformed mixture, and pour the mixture into a 1.5ml centrifuge tube. Let stand at 30℃ for 1-2h. Take 500μL of the transformed bacterial solution and spread it on YPD medium containing G418100μg / ml and D-sorbitol (1M), and culture it upside down in an incubator at 30℃ for 3 days. Extract the plasmid from the electroporated Saccharomyces cerevisiae. The recombinant strain containing the recombinant plasmid of the site-saturated mutation gene library and the pYES2NTc-spt15 plasmid is screened for transformants using a screening medium lacking uracil to obtain the desired recombinant Saccharomyces cerevisiae strain.

[0100] 7. Construction of control recombinant strains

[0101] The genome of Saccharomyces cerevisiae INVSC1 was extracted using a genome extraction kit.

[0102] Primers G418-R and G418-F were designed based on the known G418 marker gene sequence number. With reference to the known enzyme cleavage map, a restriction site was added to the forward primer and a NotⅠ restriction site was added to the primer. The primers are as follows: G418-F5'-CGGCTCCAGCCGCGCCAGATTAGC-3' (SEQ ID NO: 14)

[0103] G418-R 5'-CCCATATGAGCTCGTTTTCGACACTGGA-3' (SEQ ID NO: 15).

[0104] The control strain was constructed according to the recombinant strain construction method of pYES2NTc-spt15 plasmid. The control recombinant strain was identified by PCR method, and the G418 marker gene was amplified by G418-F and G418-R primers.

[0105] result

[0106] 1. Cloning of target genes and construction of recombinant plasmids

[0107] The genomic DNA of S.cerevisiae INVSC1 was used as a template and the designed primers were used for PCR reaction. The electrophoresis of the PCR product showed an obvious band at around 0.75 kb ( Figure 2 ), which is consistent with the expected target fragment size, and there is no specific product. The spt15 gene after purification and recovery of the PCR product was connected with the cloning vector pMD18-T, and the ligation product was transformed into E. coli DH5α. The positive transformant pMD18-spt15 was screened by blue-white spot. The PCR product electrophoresis detected an obvious band at about 3kb ( Figure 2 ), the plasmid sequencing results showed that the obtained spt15 sequence was correct, and the assay report showed that the amplified fragment was the spt15 gene fragment, which had 100% homology with the gene sequence in the gene library (GenBank gene number M29459.1, protein number AAA34458.1).

[0108] The spt15 gene was amplified by PCR on the plasmid pMD18-spt15, digested by NotⅠ / BamHI, and recovered and purified by agarose gel electrophoresis. The recovered spt15 gene was connected with the pYES2NTc linear vector that was digested by NotⅠ / BamHI and recovered. The recombinant plasmid pYES2NTc-spt15 was obtained by PCR and restriction digestion verification. The verification results are as follows: Figure 2 As shown, it indicated that the expression vector pYES2NTc-spt15 was constructed correctly.

[0109] 2. Site-directed saturation gene mutation library and acquisition of recombinant Saccharomyces cerevisiae

[0110] Recombinant plasmid pYES2NTc-spt15 was used as a template for reverse overlap extension PCR. After digestion with DpnⅠ enzyme, the plasmid was self-ligated after recovery and then transformed into E. coli DH5α. Transformants were screened on kanamycin resistance plates. The spt15 gene was randomly mutated by error-prone PCR, and all pellets were verified by NotⅠ / BamHI double digestion, indicating that the mutated gene spt15 had been cloned into the vector pYES2NTc. After sequencing, the comparison of the gene sequence with the original Saccharomyces cerevisiae spt15 gene sequence showed that 12 different point mutations were obtained at lysine 127, and no mutations occurred at other sites. The results of the 12 point mutations are shown in Table 1.

[0111] The recombinant plasmid containing wild-type spt15 and G418 resistance marker genes and mutated spt15 genes was transformed into Saccharomyces cerevisiae INVSC1 by electroporation. Transformants were screened using a screening medium lacking uracil to obtain a series of recombinant Saccharomyces cerevisiae strains. Because the recombinant plasmid pYES2NTc-G418 only has a resistance marker gene and no other exogenous genes, this yeast strain was used as a control strain, INVSC1-Neo. The yeast carrying pYES2NTc-spt15 and pYES2NTc-spt3 plasmids had the wild-type spt15 gene coding region, respectively, and could overexpress the spt15 gene, and was named INVSC1-spt15 strain. The spt15 mutant genes were named a series of recombinant Saccharomyces cerevisiae INVSC1-spt15-X according to the sequence number, where X is F, V, R, M, L, G, T, S, Q, D, N, I (see Table 1).

[0112] Table 1 Codons and amino acids at site-directed saturation mutagenesis sites

[0113]

[0114]

[0115] Example 2

[0116] Method for screening tolerance of recombinant Saccharomyces cerevisiae

[0117] 1. Screening of acetic acid-tolerant recombinant yeast strains

[0118] The acetic acid concentration gradient was set to 4g / L, 5g / L, 6g / L, and 8g / L. First, the YPD medium was sterilized at high temperature. When it was cooled to about 50°C, a certain amount of acetic acid was added according to the set gradient. Then the control group strains were streaked and inoculated into the above-mentioned gradient plates, the plates were inverted, and grown in the incubator at 30°C for 2 days to observe the growth. The growth of the strains in the incubator was judged. The concentration at which the control strain could not grow on the plate containing acetic acid was the highest acetic acid resistance concentration. With this concentration as the standard, plates were made, and the recombinant strains were streaked and inoculated into the plates, and cultured at 30°C for 2 days to screen the recombinant strains that could grow on the plates at this concentration. The acetic acid concentration was increased successively until the recombinant strain with the highest acetic acid resistance concentration was screened. After the screened acetic acid-resistant recombinant strains and control strains were activated and cultured on the YPD slant, they were inoculated into the seed culture medium respectively and cultured overnight at 30°C and 200r / min. The concentration of bacterial solution was adjusted to keep the inoculation amount consistent, and the culture medium containing acetic acid (the highest concentration of acetic acid resistance) and 100 mL of acetic acid-free fermentation medium was inoculated, and the culture was carried out at 30°C and 200 r / min. During the fermentation process, a certain amount of fermentation liquid was taken at intervals, and the OD value was measured at 600 nm by a spectrophotometer after dilution, and the growth was compared. Compared with the control strain containing an empty vector, the mutant with improved acetic acid tolerance grew well in the fermentation medium with the highest concentration of acetic acid resistance. The experiment was carried out three times, and the conditions were kept the same each time, and all the results were repeatable.

[0119] 2. Screening of hyperosmotic-tolerant recombinant yeast strains

[0120] The NaCl gradient was set to 6%, 7%, and 8%. A certain amount of NaCl was added to the YPD medium according to the designed gradient. After the medium was sterilized at high temperature, the control group strains were streaked and inoculated into the above four gradient plates. The plates with different gradients were inverted and cultured in the incubator. The temperature was set to 30°C. The strains were grown for 2 days under this condition, and the growth of the strains on the plates was observed. According to the growth in different gradient plates, the concentration at which the control strain could not grow in the NaCl plate was the highest concentration of NaCl resistance. With this concentration as the standard, plates were made, and the recombinant strains were streaked and inoculated into the plates, cultured at 30°C for 2 days, and strains that could grow in the plates at this concentration were screened. The NaCl concentration was increased successively until the recombinant strain with the highest NaCl resistance was screened. After the selected NaCl-resistant recombinant strains and control strains were activated and cultured on the YPD slope, they were inoculated into the seed culture medium and cultured overnight at 30°C and 200r / min. The bacterial liquid concentration was adjusted to keep the inoculation amount consistent, and the culture medium was inoculated into 100 mL of fermentation medium containing NaCl (highest NaCl concentration resistant) and excluding NaCl. The culture medium was fermented at 30°C and 200 r / min for 48 h. During the fermentation process, a certain amount of fermentation liquid was taken at regular intervals, diluted, and its OD value was measured at 600 nm with a spectrophotometer to compare its growth. Compared with the control strain containing an empty vector, the mutant that grew well in the fermentation medium resistant to the highest NaCl concentration was a mutant with improved hyperosmotic tolerance.

[0121] 3. Screening of thermotolerant recombinant yeast strains

[0122] The control group strain was streaked and inoculated in the YPD culture after high temperature sterilization, and then the culture medium was placed in the incubator at 37℃, 40℃, and 42℃ for 2 days to observe its growth. According to the growth of the strain in the incubator at different temperatures, the highest temperature at which it cannot grow in the incubator is the highest tolerance temperature. Using this temperature as a standard, the recombinant strains were streaked and inoculated into the plate, and the incubator temperature was increased successively until the recombinant strain with the highest high temperature resistance was screened out. After the selected high temperature resistant recombinant strains and control strains were activated and cultured on the YPD slant, they were inoculated into the seed culture medium and cultured overnight at 30℃ and 200r / min. The bacterial liquid concentration was adjusted to keep the inoculation amount consistent, and the culture medium containing 100 mL was inoculated. Under the conditions of the highest temperature and 30°C, the culture medium was fermented at a speed of 200 r / min for 48 h. Under the fermentation conditions of different temperatures, a certain amount of fermentation liquid was taken at regular intervals, and the taken fermentation liquid was diluted. The OD value was measured at 600 nm with a spectrophotometer, and the growth of each strain under different conditions was compared. Compared with the control strain containing an empty vector, the mutant that grew well in the fermentation medium that was resistant to the highest temperature was a mutant with improved temperature tolerance.

[0123] 4. Screening of ethanol-tolerant recombinant yeast strains

[0124] The gradient of ethanol addition was set to 13%, 15%, and 17%. After the YPD medium was sterilized at high temperature, a certain amount of ethanol was added when it cooled to room temperature. Plates were made separately, and the control group strains were streaked and inoculated into the above three gradient plates. The plates containing different ethanol gradients were inverted into the incubator, cultured at 30°C for 2 days, and their growth was observed. According to the growth of the strains in different ethanol gradient plates, the highest ethanol tolerance concentration of the control strain was determined. With this concentration as the standard, plates were made, and the recombinant strains were streaked and inoculated into the plates, cultured at 30°C for 2 days, and the recombinant strains that could grow in the plates at this concentration were screened. The ethanol concentration was increased successively until the recombinant strain with the highest ethanol tolerance concentration was screened. After the screened ethanol-resistant recombinant strains and control strains were activated and cultured on the YPD slant, they were inoculated into the seed culture medium respectively, and cultured overnight at 30°C and 200r / min. The bacterial liquid concentration was adjusted to keep the inoculation amount consistent, and the culture medium was inoculated into 100 mL of the fermentation medium with the highest ethanol tolerance volume fraction and without ethanol. The culture medium was fermented at 30°C and 200 r / min for 48 h. During the fermentation process, a certain amount of fermentation liquid was taken at regular intervals, diluted, and its OD value was measured at 600 nm with a spectrophotometer. Compared with the control strain containing an empty vector, the mutant that grew well in the fermentation medium with the highest ethanol tolerance volume fraction was a mutant with improved ethanol tolerance.

[0125] result

[0126] 1. Screening of acetic acid-tolerant recombinant yeast strains

[0127] 1.1 Preliminary screening of acetic acid-resistant recombinant strains

[0128] The control strain was inoculated into YPD medium with acetic acid concentrations of 4, 5, 6, and 7 g / L to test its acetic acid tolerance. Figure 3 As shown in Figure A, the control strain INVSC1-Neo can grow in a medium with a content of 5 g / L, but when the acetic acid content in the plate is greater than 6 g / L, the control strain can hardly grow, indicating that the highest concentration of acetic acid that the control strain INVSC1-Neo can tolerate is 5 g / L. The mutant recombinant strains can grow in a medium with a content of 5 g / L, but when inoculated into a medium with a content of 6 g / L acetic acid, Figure 3 As shown in B, only INVSC1-spt15-G and INVSC1-spt15-Q strains can grow, while other recombinant yeast strains and recombinant yeast strains cannot grow. Therefore, the experiment shows that these two strains have acquired a certain degree of acetic acid tolerance, and the highest acetic acid concentration that can be tolerated is 6g / L.

[0129] 1.2 Rescreening of acetic acid-resistant recombinant strains

[0130] The INVSC1-spt15-G, INVSC1-spt15-Q and INVSC1-Neo control strains screened on the plate were inoculated into the seed culture medium and cultured overnight at 30°C and 200 r / min. The bacterial solution concentration was adjusted to keep the inoculum consistent, and the culture medium with an acetic acid concentration of 6 g / L and without acetic acid was inoculated, and the OD value was measured at 600 nm using a spectrophotometer.

[0131] The results are shown in Table 2. As can be seen from the table, the OD values ​​of the recombinant strains INVSC1-spt15-G and INVSC1-spt15-Q and the control strain INVSC1-Neo increased most significantly in the logarithmic phase, and the growth of each strain was inconsistent under different growth conditions.

[0132] Table 2 OD values ​​of recombinant strains in acetic acid-containing medium

[0133]

[0134]

[0135] According to the test results, the tolerance growth curve of the acetic acid-resistant recombinant yeast strain was obtained with time as the horizontal axis and OD value as the vertical axis, as shown in Figure 4 shown.

[0136] The tolerance growth curve of yeast under acetic acid stress showed that INVSC1-spt15-G and INVSC1-spt15-Q could tolerate acetic acid at a concentration of 6 g / L and grew well under the condition of acetic acid concentration of 6 g / L, while the INVSC1-Neo control strain could not grow under the condition of acetic acid concentration of 6 g / L and the OD value remained basically unchanged, which was consistent with the results of the initial plate screening. The growth of INVSC1-spt15-G, INVSC1-spt15-Q and INVSC1-Neo control strains under the condition of no acid addition was significantly better than that under the condition of acid addition. Figure 4 It can be seen that when the medium was added with acetic acid at a concentration of 6 g / L, the acetic acid tolerance of INVSC1-spt15-G and INVSC1-spt15-Q was 3.64 times and 4.79 times that of INVSC1-Neo after 48 hours of culture, and the tolerance of INVSC1-spt15-Q to acetic acid was slightly higher than that of INVSC1-spt15-G. This indicates that INVSC1-spt15-G and INVSC1-spt15-Q have acquired a certain degree of tolerance under the condition of an acetic acid concentration of 6 g / L. Therefore, through primary screening and rescreening, the two recombinant strains INVSC1-spt15-G and INVSC1-spt15-Q were finally selected, and the highest acetic acid concentration resistant was 6 g / L.

[0137] 2. Screening of recombinant yeast strains resistant to hyperosmotic pressure (NaCl)

[0138] 2.1 Preliminary screening of recombinant strains resistant to hyperosmotic pressure (NaCl)

[0139] The control strain was inoculated into culture medium with NaCl content of 6%, 7%, 8%, and 9% to test its sodium chloride tolerance. Figure 5 As shown in A, the control strain INVSC1-Neo can grow in a culture medium with a NaCl mass fraction of 7%, but at this time, there are basically no colonies at the end of the line of the control strain INVSC1-Neo, and it cannot grow under conditions where the NaCl mass fraction is greater than 7%, indicating that the highest concentration of sodium chloride that the control strain INVSC1-Neo can tolerate is 7%. The mutant recombinant strain can grow in a culture medium with a NaCl mass fraction of 7%. When inoculated into a culture medium with a NaCl mass fraction of 9%, as shown in Figure 5 As shown in B, only INVSC1-spt15-G and INVSC1-spt15-S strains can grow, while other recombinant yeast strains and control strains cannot grow. Therefore, the experiment shows that these two strains have acquired a certain degree of high osmotic tolerance and can tolerate 9% NaCl by mass.

[0140] 2.2 Rescreening of recombinant strains resistant to hyperosmotic pressure (NaCl)

[0141] The INVSC1-spt15-G, INVSC1-spt15-S and INVSC1-Neo control strains screened on the plate were inoculated into the seed culture medium and cultured overnight at 30°C and 200r / min. The bacterial liquid concentration was adjusted to keep the inoculation amount consistent, and they were inoculated into the fermentation medium with a NaCl mass fraction of 9% and without NaCl, respectively. Samples were taken at a certain time and their OD values ​​at 600nm were measured.

[0142] The results are shown in Table 3. As can be seen from Table 3, the OD values ​​of INVSC1-spt15-G, INVSC1-spt15-S and INVSC1-Neo control strains in 0 and 9% NaCl mass fraction YPD medium are inconsistent, and the OD values ​​of the recombinant strains are most different in the logarithmic phase.

[0143] Table 3 OD values ​​of recombinant strains in NaCl-containing medium

[0144]

[0145]

[0146] According to the measurement results, the tolerance growth curve of the hyperosmotic recombinant yeast strain was obtained with time as the horizontal axis and OD value as the vertical axis, as shown in Figure 6 shown.

[0147] Tolerance growth curve of recombinant yeast under NaCl stress ( Figure 6 ) It can be seen that the recombinant strains INVSC1-spt15-G and INVSC1-spt15-S grow well in a medium with a sodium chloride mass fraction of 9%, and can tolerate 9% sodium chloride. Under this condition, their growth is significantly better than that of the INVSC1-Neo control strain. This result is no different from the results of the initial plate screening, both of which show that INVSC1-spt15-G and INVSC1-spt15-S can grow under the condition of a sodium chloride mass fraction of 9%. Figure 3-5 It can be seen that when the culture medium was added with 9% NaCl mass fraction, the tolerance of the recombinant strains INVSC1-spt15-G and INVSC1-spt15-S to NaCl was 1.16 times and 1.75 times that of the INVSC1-Neo control strain, respectively, after culturing for 48 hours. The growth of INVSC1-spt15-G, INVSC1-spt15-S and INVSC1-Neo control strains without NaCl was better than that with NaCl, indicating that the recombinant strains INVSC1-spt15-G and INVSC1-spt15-S had a certain tolerance under the condition of 9% sodium chloride mass fraction.

[0148] 3. Screening of thermotolerant recombinant yeast strains

[0149] 3.1 Preliminary screening of thermostable recombinant yeast strains

[0150] The control strain was cultured at 35°C and the temperature gradient was gradually increased until the culture temperature reached 41°C. Figure 7 As shown in A, the control bacteria INVSC1-Neo can grow under this condition, but cannot grow when the incubator temperature is greater than 41°C. Therefore, the high temperature resistance condition of the control bacteria INVSC1-Neo is determined to be 41°C. Taking the culture temperature of 41°C as the standard, the culture temperature is increased in sequence, such as Figure 7 As shown in B, only INVSC1-spt15-I and INVSC1-spt15-M can grow under a culture temperature of 43°C, while other strains cannot grow under this condition. Therefore, the experiment shows that INVSC1-spt15-I has the strongest temperature tolerance among the recombinant strains, with a maximum tolerance temperature of 43°C, and cannot grow above this temperature. Through initial plate screening, two high-temperature resistant recombinant yeast strains, INVSC1-spt15-I, were obtained, and the highest temperature they can tolerate is 43°C.

[0151] 3.2 Rescreening of thermostable recombinant yeast strains

[0152] INVSC1-spt15-I and INVSC1-spt15-M screened on the plate and the control strain INVSC1-Neo were inoculated into the seed culture medium and cultured overnight at 30°C and 200r / min. The OD values ​​of the fermentation culture at different temperatures were measured. The results showed that the OD values ​​of INVSC1-spt15-I, INVSC1-spt15-M and the control strain INVSC1-Neo had no difference under the culture condition of 43°C, which was different from the results of the plate screening. This shows that when the fermentation medium temperature is 43°C, compared with the control strain INVSC1-NeoI, INVSC1-spt15-I and INVSC1-spt15-M do not show the superiority of high temperature resistance.

[0153] The other conditions remained unchanged, and the culture conditions were set to 42°C and 30°C, respectively, and cultured in the fermentation medium at a rotation speed of 200 r / min.

[0154] The results are shown in Table 4. It can be seen from Table 4 that the OD values ​​of INVSC1-spt15-I, INVSC1-spt15-M and the control strain INVSC1-Neo in the culture medium at 30°C and 42°C all increased with time, but there were obvious differences in the growth conditions, especially in the logarithmic phase.

[0155] Table 4 OD values ​​of recombinant strains in culture medium at different temperatures

[0156]

[0157]

[0158] According to the measurement results, the tolerance growth curve of the high temperature resistant recombinant yeast strain was obtained with time as the horizontal axis and OD value as the vertical axis, as shown in Figure 8 shown.

[0159] Tolerance growth curve of recombinant yeast under high temperature stress ( Figure 8 ) can be seen from the figure that in the YPD medium at 30°C, the growth of INVSC1-spt15-I, INVSC1-spt15-M and the control strain INVSC1-Neo was significantly better than that in the medium at 42°C, and there was no significant difference in their growth. Figure 8It is shown in the figure that the recombinant strains INVSC1-spt15-I and INVSC1-spt15-M can tolerate a high temperature of 42°C, that is, the recombinant strains INVSC1-spt15-I and INVSC1-spt15-M can grow under these conditions, while the control strain INVSC1-Neo cannot grow under the high temperature of 42°C. Figure 8 The results show that when the fermentation medium temperature is 42°C, the tolerance of INVSC1-spt15-I and INVSC1-spt15-M is 1.58 times and 1.45 times that of the control strain INVSC1-Neo, respectively. Through primary and secondary screening experiments, the high-temperature resistant recombinant strains INVSC1-spt15-I and INVSC1-spt15-M were finally selected, and the highest temperature they can tolerate is 42°C.

[0160] 4. Screening of ethanol-tolerant recombinant yeast strains

[0161] 4.1 Preliminary screening of ethanol-tolerant recombinant yeast strains

[0162] The control strain INVSC1-Neo was inoculated into YPD medium with ethanol volume fractions of 12%, 14%, and 16% respectively to test its ethanol tolerance. The results showed that only the growth of each strain in the 14% and 16% ethanol concentration groups showed significant differences. Fig. 9 As shown in Figure A, the control bacteria can grow in a medium with a volume fraction of 14% ethanol. When inoculated into a medium with a volume fraction of 16% ethanol, Fig. 9 As shown in B and C, only the recombinant strains INVSC1-spt15-G, INVSC1-spt15-S and INVSC1-spt15-D can grow, so this experiment shows that only these three strains have acquired a certain degree of ethanol tolerance.

[0163] 4.2 Rescreening of ethanol-tolerant recombinant yeast strains

[0164] After seed culture, the selected strains were inoculated into fermentation medium with 16% ethanol volume fraction and without ethanol, and the OD values ​​were measured by spectrophotometer. The results are shown in Table 5. As can be seen from Table 5, the OD values ​​of INVSC1-spt15-G, INVSC1-spt15-S, INVSC1-spt15-D and INVSC1-Neo in YPD medium with 0% and 16% ethanol volume fraction increased with time, but the growth conditions were different, especially in the logarithmic phase.

[0165] Table 5 OD values ​​of recombinant strains in ethanol-containing medium

[0166]

[0167] According to the measurement results, the tolerance growth curve of the ethanol-tolerant recombinant yeast strain was obtained with time as the horizontal axis and OD value as the vertical axis, as shown in Fig.10 shown.

[0168] From the tolerance growth curve of recombinant yeast under ethanol stress ( Fig.10 ) It can be seen that the recombinant strains INVSC1-spt15-G, INVSC1-spt15-S, and INVSC1-spt15-D can tolerate 16% ethanol by volume. When the medium is added with 16% ethanol by volume, after culturing for 48 hours, the ethanol tolerance of INVSC1-spt15-G, INVSC1-spt15-S, and INVSC1-spt15-D is 2.76 times, 4.17 times, and 3.08 times that of the INVSC1-Neo control group, respectively. The strains arranged from high to low in terms of tolerance are: INVSC1-spt15-S, INVSC1-spt15-D, and INVSC1-spt15-G. It can be more clearly seen from the tolerance curve that the growth of INVSC1-spt15-G, INVSC1-spt15-S, and INVSC1-spt15-D in high volume fraction ethanol culture medium is significantly lower than that in culture medium without ethanol.

[0169] The spt15 site-saturated mutant gene of Saccharomyces cerevisiae was obtained by genetic engineering and connected to the expression vector pYES2NTc to construct a mutant library. After sequencing, a total of 12 single-point mutant genes were obtained. The recombinant plasmids containing the 12 point mutant genes and the wild-type genes spt15, spt3 and the control gene Neo were transferred into Saccharomyces cerevisiae INVSC1 by electroporation, and 15 recombinant Saccharomyces cerevisiae strains were obtained.

[0170] Through primary and secondary screening of recombinant yeast strains under conditions of resistance to acetic acid, hyperosmotic pressure, high temperature and ethanol, two mutant genes, spt15-G and spt15-Q, were obtained, which can improve the yeast's resistance to acetic acid; two mutant genes, spt15-G and spt15-S, were obtained, which can improve the yeast's resistance to sodium chloride; two mutant genes, spt15-I and spt15-M, were obtained, which can improve the yeast's resistance to high temperature; three mutant genes, spt15-G, spt15-S and spt15-D, were obtained, which can improve the brewer's resistance to ethanol.

[0171] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a yeast initiation transcription factor mutant protein, a gene encoding the yeast initiation transcription factor mutant protein, or a genetic modification product comprising the gene encoding the gene in industrial production of ethanol; The yeast initiation transcription factor mutant protein is obtained by mutation of the 127th lysine of the yeast initiation transcription factor spt15 to one of the following amino acid residues: glycine, glutamine, serine, isoleucine and aspartic acid.

2. The use according to claim 1, It is characterized in that The amino acid sequence of the yeast initiation transcription factor spt15 is shown in SEQ ID NO: 1; Preferably, when mutated to glycine, the yeast initiation transcription factor mutant protein is spt15-G, and the amino acid sequence is shown in SEQ ID NO: 2; When mutated to glutamine, the yeast initiation transcription factor mutant protein is spt15-Q, and the amino acid sequence is shown in SEQ ID NO: 3; When mutated to serine, the yeast initiation transcription factor mutant protein is spt15-S, and the amino acid sequence is shown in SEQ ID NO:4; When mutated to isoleucine, the yeast initiation transcription factor mutant protein is spt15-I, and the amino acid sequence is shown in SEQ ID NO: 5; When mutated to aspartic acid, the yeast initiation transcription factor mutant protein is spt15-D, and the amino acid sequence is shown in SEQ ID NO:

6.

3. The use according to claim 2, It is characterized in that The nucleotide sequences of the genes encoding the yeast initiation transcription factor mutant proteins spt15-G, spt15-Q, spt15-S, spt15-I and spt15-D are shown in at least one of SEQ ID NO: 16 to SEQ ID NO:

20.

4. The use according to claim 3, It is characterized in that The genetic modification product containing the encoding gene includes a recombinant vector containing the encoding gene, a recombinant yeast strain containing the encoding gene, or a recombinant yeast strain containing the recombinant vector.

5. The use according to claim 3, It is characterized in that When the yeast initiation transcription factor mutant protein is spt15-G or spt15-Q, during the process of producing industrial ethanol by the recombinant yeast strain, the acetic acid concentration in the fermentation system is 5-6 g / L.

6. The use according to claim 3, It is characterized in that When the yeast initiation transcription factor mutant protein is spt15-G or spt15-S, during the process of producing industrial ethanol by the recombinant yeast strain, the mass percentage of sodium chloride in the fermentation system is 7% to 9%.

7. The use according to claim 3, It is characterized in that When the yeast initiation transcription factor mutant protein is spt15-I or spt15-S, during the process of producing industrial ethanol by the recombinant yeast strain, the fermentation temperature is 41-43°C.

8. The use according to claim 3, It is characterized in that When the yeast initiation transcription factor mutant protein is spt15-G, spt15-S or spt15-D, during the process of producing industrial ethanol by the recombinant yeast strain, the volume percentage of ethanol in the fermentation system is 14% to 19%.

9. Use of a yeast initiation transcription factor mutant protein, a gene encoding the yeast initiation transcription factor mutant protein, or a recombinant vector comprising the gene encoding the yeast initiation transcription factor mutant protein in improving yeast tolerance to a fermentation environment, wherein the yeast initiation transcription factor mutant protein is the yeast initiation transcription factor mutant protein in the use according to claim 1 or 2.

10. The use according to claim 9, It is characterized in that When the yeast initiation transcription factor mutant protein is spt15-G or spt15-Q, use of the yeast initiation transcription factor mutant protein, the encoding gene of the yeast initiation transcription factor mutant protein or the recombinant vector in improving yeast tolerance to acetic acid; When the yeast initiation transcription factor mutant protein is spt15-G or spt15-S, use of the yeast initiation transcription factor mutant protein, the encoding gene of the yeast initiation transcription factor mutant protein or the recombinant vector in improving yeast tolerance to sodium chloride; When the yeast initiation transcription factor mutant protein is spt15-I or spt15-S, use of the yeast initiation transcription factor mutant protein, the encoding gene of the yeast initiation transcription factor mutant protein or the recombinant vector in improving yeast tolerance to fermentation temperature; When the yeast initiation transcription factor mutant protein is spt15-G, spt15-S or spt15-D, the yeast initiation transcription factor mutant protein, the encoding gene of the yeast initiation transcription factor mutant protein or the recombinant vector are used in improving yeast tolerance to ethanol.

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