Method for enhancing efficiency of producing ethanol from saccharomyces cerevisiae

Abstract

A method for enhancing the efficiency of producing ethanol from Saccharomyces cerevisiae, comprising: integrally expressing lectin protein in a Saccharomyces cerevisiae genome to obtain recombinant Saccharomyces cerevisiae, and producing ethanol from the recombinant Saccharomyces cerevisiae in a fermentation system containing a solid carrier. In the surface adhesion continuous production of ethanol, the constructed recombinant Saccharomyces cerevisiae strain can promote the adhesion and growth of yeast cells on a solid medium by improving the adhesion and membrane-forming capability of the yeast cells, so that a continuous production process is achieved, the population growth density of the cells can also be improved, the tolerance of the cells and the resistance thereof to adverse production environments can be enhanced, and the reaction properties of the yeast cells and the production efficiency of ethanol are improved. Additionally, in the continuous production of ethanol by coupling surface adhesion fermentation and membrane separation, the turbidity of a fermentation liquor can be reduced, the pollution to a separated membrane can be reduced, the efficiency of membrane separation can be improved, and the fermentation metabolic byproducts, glycerol and succinic acid, can be reduced.

Description

A method for enhancing the efficiency of ethanol production by brewer's yeast Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a method for enhancing the efficiency of ethanol production by brewer's yeast and a method for continuously producing ethanol by coupling surface adhesion fermentation with membrane separation. Background Art

[0002] Saccharomyces cerevisiae is a generally recognized as safe (GRAS) eukaryotic model microorganism characterized by simple cultivation, rapid cell growth, a well-defined physiological and genetic background, and a comprehensive suite of genetic manipulation tools. Due to its anaerobic metabolism of glucose and high sugar alcohol conversion efficiency, Saccharomyces cerevisiae is a preferred microorganism for bioethanol production. Currently, mainstream bioethanol fermentation relies primarily on free-cell, batch fermentation, requiring repeated breeding, inoculation, tank washing, and sterilization steps, resulting in low efficiency and high costs. Research reports indicate that biofilm cells exhibit self-replication, self-repair, and strong environmental tolerance. Therefore, surface-adherent fermentation systems constructed based on biofilms can achieve continuous ethanol production. The flocculant proteins encoded by the FLO family genes in Saccharomyces cerevisiae play a crucial role in biofilm formation. The proteins encoded by the FLO1, FLO5, FLO9, and FLO10 genes promote cell-to-cell adhesion, while the protein encoded by the FLO11 gene promotes cell-to-surface and cell-to-cell adhesion. The flocculant proteins encoded by the ALS family genes in Candida albicans also have the ability to promote biofilm formation. Currently, there are numerous reports on the use of plasmids to express FLO and ALS family genes in Saccharomyces cerevisiae to study the phenotypes of these genes. However, in industrial applications, recombinant strains expressing target genes using episomal plasmids have difficulty achieving long-term continuous fermentation due to plasmid loss. In summary, the present invention provides a method for enhancing the efficiency of ethanol production in Saccharomyces cerevisiae and a method for continuous ethanol production by coupling surface-adhesive fermentation with membrane separation.

[0003] Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for enhancing the efficiency of ethanol production by brewer's yeast in view of the shortcomings of the existing technology.

[0005] Inventive concept: FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3, and ALS5 genes have the ability to promote cell-to-cell adhesion or cell-to-interface adhesion. The present invention integrates and expresses any one or a combination of FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3, and ALS5 genes in the Saccharomyces cerevisiae genome to construct a recombinant Saccharomyces cerevisiae strain, thereby promoting the adhesion ability between yeast cells or between cells and interfaces, and ultimately improving the surface adhesion ability of the recombinant strain. In a surface adhesion continuous fermentation system, the surface adhesion performance of yeast cells is enhanced, the in situ replacement of living and dead cells is achieved, and the production efficiency of ethanol is enhanced. Unlike traditional encapsulation immobilization fermentation, cross-linking immobilization fermentation and other methods, yeast cells in surface adhesion fermentation are self-adsorbed on solid carriers, and the in situ replacement of living and dead cells can be achieved, thereby having better cell activity and mass transfer, heat transfer and other properties.

[0006] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0007] In a first aspect, the present invention discloses a method for enhancing the ethanol production efficiency of Saccharomyces cerevisiae, wherein a lectin protein is integrated and expressed in the genome of Saccharomyces cerevisiae to obtain recombinant Saccharomyces cerevisiae, and the recombinant Saccharomyces cerevisiae produces ethanol in a fermentation system containing a solid carrier.

[0008] Wherein, the lectin protein includes any one or several combinations of FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3 and ALS5, such as single-copy expression of genes such as FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3, ALS5, such as the combination of FLO11-FLO11, double-copy expression of single genes such as the combination of FLO9-FLO11, the combination of FLO10-ALS3, the combination of FLO11-ALS3, the combination of ALS3-ALS5, the combination of FLO9-FLO10-FLO11, the combination of FLO10-FLO11-ALS3, the combination of FLO5-FLO11, the combination of FLO5-FLO10-FLO11, the combination of FLO1-FLO9-ALS3, the combination of FLO1-FLO10, or the combination of FLO10-ALS1-ALS3, etc.

[0009] The accession numbers of the amino acid sequences of FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3 and ALS5 are NP_009424.1, NP_012081.1, NP_009338.1, NP_013028.1, NP_012284.3, XP_718077.1, XP_710435.2, XP_71807 4.2; Preferably, the registration numbers of the nucleotide sequences of FLO1, FLO5, FLO9, FLO10, and FLO11 are NM_001178230.1, NM_001179342.1, NM_001178205.1, NM_001179892.1, and NM_001179541.3, respectively; the nucleotide sequences of ALS1, ALS3, and ALS5 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0010] The brewer's yeast is any one of Saccharomyces cerevisiae S288C and Saccharomyces cerevisiae CICC1308; preferably, the brewer's yeast is any one of Saccharomyces cerevisiae BY4742, Saccharomyces cerevisiae W303-1A and Saccharomyces cerevisiae CICC1308.

[0011] Among them, there are many ways of integration and expression, including homologous recombination, CRISPR gene editing technology, such as CRISPR / Cas9 gene editing technology.

[0012] The method for constructing the recombinant Saccharomyces cerevisiae is to integrate and express any one or a combination of several of the FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3 and ALS5 lectin protein genes into Saccharomyces cerevisiae.

[0013] The integrated expression into Saccharomyces cerevisiae is integrated expression into any one of Saccharomyces cerevisiae chromosomes 1-16; preferably, the integrated expression is into Saccharomyces cerevisiae chromosomes 1, 4, or 16; further preferably, the integrated expression is into Saccharomyces cerevisiae chromosomes 1, 4, or 16, and the insertion site of the integrated expression is 106a, 416d, or 1622b.

[0014] Specifically, the construction of the recombinant Saccharomyces cerevisiae strain includes the following steps:

[0015] (1) Using CRISPR / Cas9 gene editing technology, the 20-nt sequence in the plasmid is replaced with the sequence of the gene integration site to obtain a reconstructed CRISPR / Cas9 gene editing plasmid, such as pCAS-1622b, pCAS-106a, and pCAS-416d;

[0016] (2) Extracting the genome of Saccharomyces cerevisiae;

[0017] (3) Using the genome of Saccharomyces cerevisiae as a template, the upstream homology arm sequence, downstream homology arm sequence, promoter sequence, target gene sequence, and terminator sequence were obtained by PCR;

[0018] (4) connecting the gene fragments described in (3) in the order of upstream homologous arm-promoter-target gene-terminator-downstream homologous arm to obtain homologous recombinant fragments;

[0019] (5) The reconstructed CRISPR / Cas9 gene editing plasmid and the homologous recombination fragment described in (1) and (4) are transferred into Saccharomyces cerevisiae to obtain a recombinant Saccharomyces cerevisiae strain.

[0020] In step (1), the plasmid is a pCAS plasmid.

[0021] In step (1), the 20-nt sequence (1622b) of the gene integration site in chromosome 16 is shown as SEQ ID NO.4; the 20-nt sequence (106a) of the gene integration site in chromosome 1 is shown as SEQ ID NO.5; and the 20-nt sequence (416d) of the gene integration site in chromosome 4 is shown as SEQ ID NO.6.

[0022] In step (3), the promoter used is a constitutive promoter; preferably, the promoter used in the integrated expression is TPI or any one of TEF1 and TDH3; preferably, the terminator used in the integrated expression is CYC1 or ADH1.

[0023] In step (3), the target genes FLO1, FLO5, FLO9, FLO10, and FLO11 were obtained by PCR using the genome of Saccharomyces cerevisiae S288C, Saccharomyces cerevisiae BY4742, Saccharomyces cerevisiae W303-1A, or Saccharomyces cerevisiae CICC1308 as templates; the target genes ALS1, ALS3, and ALS5 were synthesized by a biotechnology company.

[0024] In step (5), the recombinant Saccharomyces cerevisiae strain is screened in a YPD medium containing 100-600 mg / L G418 antibiotic. Preferably, the recombinant Saccharomyces cerevisiae strain is screened in a YPD medium containing 500 mg / L G418 antibiotic.

[0025] The first-level seed liquid of the recombinant Saccharomyces cerevisiae is inoculated into the culture medium at a volume ratio of 1%-50%, preferably 5%-15%, and more preferably 10%, for surface adhesion culture; the culture medium is the second-level seed liquid.

[0026] The solid carrier is any one of glass sheet, cotton fiber, polyester fiber, nylon fiber, wood pulp cotton, polylactic acid, activated carbon, polyethylene, polyvinyl alcohol, polyurethane, clay, metal and ceramic, preferably cotton fiber; preferably, the amount of the solid carrier is 1-100 g / L culture medium, preferably 40-60 g / L culture medium, further preferably 40 g / L culture medium; the culture medium is a secondary seed solution.

[0027] The content of glucose in the fermentation medium used in the fermentation process is 5-300 g / L, preferably 60-250 g / L, and more preferably 200 g / L; preferably, the fermentation medium further comprises tryptone, yeast powder, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate, zinc sulfate heptahydrate, and ferrous sulfate heptahydrate; preferably, the concentration of each component in the fermentation medium except glucose is tryptone 2-6 g / L, yeast powder 1-5 g / L, potassium dihydrogen phosphate 1-5 g / L, ammonium sulfate 2-6 g / L, magnesium sulfate 0.3-0.7 g / L, zinc sulfate heptahydrate 0.03-0.07 g / L, ferrous sulfate heptahydrate 0.03-0.07 g / L, preferably 3-5 g / L tryptone, 2-4 g / L yeast powder, 2-4 g / L potassium dihydrogen phosphate, 3-5 g / L ammonium sulfate, 0.4-0.6 g / L magnesium sulfate, 0.04-0.06 g / L zinc sulfate heptahydrate, 0.04-0.06 g / L ferrous sulfate heptahydrate, more preferably 4 g / L tryptone, 3 g / L yeast powder, 3 g / L potassium dihydrogen phosphate, 4 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 0.05 g / L zinc sulfate heptahydrate, 0.05 g / L ferrous sulfate heptahydrate, and the solvent is water.

[0028] The recombinant Saccharomyces cerevisiae produces ethanol by adherence fermentation in a fermentation system containing a solid carrier; the surface adherence fermentation refers to producing ethanol in a fermentation system containing yeast cells and a solid carrier for yeast adhesion in a reactor.

[0029] Wherein, the adhesion fermentation is surface adhesion continuous fermentation.

[0030] Among them, the surface adhesion continuous fermentation is to add a solid carrier to the seed culture medium or fermentation culture medium at the initial stage of fermentation for fermentation. After each batch of fermentation is completed, the fermentation liquid is released and the solid carrier is retained, and fresh fermentation culture medium is added to start the next batch of fermentation culture, and this cycle is repeated until the fermentation is completed; among them, the initial stage of fermentation is before the seed liquid is inoculated, specifically before the first-level seed liquid is connected to the second-level seed liquid.

[0031] Preferably, the adding of the solid carrier to the seed culture medium or the fermentation medium for fermentation at the initial stage of fermentation is to inoculate the first seed liquid into the second seed liquid containing the solid carrier for surface adhesion culture.

[0032] Preferably, the surface adhesion continuous fermentation is to connect the first-level seed liquid to the second-level seed liquid containing the solid carrier for static culture first, and then flow circulation culture. After the surface adhesion fermentation is completed, the second-level seed liquid is released and the solid carrier is retained, and fresh fermentation medium is added to start fermentation culture, and this cycle is repeated until the fermentation is completed.

[0033] Wherein, the temperature of the static culture and the flow circulation culture is 25-35°C, preferably 28-32°C, and more preferably 30°C; preferably, the static culture time is 1-7h, preferably 3-5h, and more preferably 4h; preferably, the flow circulation culture rate is 10-50mL / min, preferably 20-40mL / min, and more preferably 30mL / min; preferably, the flow circulation culture time is 4-16h, preferably 8-12h; preferably, after the fresh fermentation medium is supplemented, the fermentation broth circulates to start the next batch of fermentation; preferably, the circulation rate of the fermentation broth is 10-150mL / min, preferably 20-40mL / min, and more preferably 30mL / min.

[0034] Wherein, when the content of glucose in the fermentation medium in the surface adhesion continuous fermentation is below 1 g / L, the batch fermentation is terminated.

[0035] The released fermentation liquid is fermentation liquid that accounts for more than 95% v / v of the total fermentation liquid, preferably all of the fermentation liquid; preferably, the volume of the fresh fermentation medium added is the same as the volume of the released fermentation liquid.

[0036] Wherein, the fermentation temperature is 25-37°C, preferably 28-37°C, and more preferably 35°C.

[0037] In the present invention, the primary seed solution is the seed solution of Saccharomyces cerevisiae or recombinant Saccharomyces cerevisiae activated in YPD medium.

[0038] In the present invention, the secondary seed liquid is a seed culture medium, which includes yeast powder, peptone, glucose, potassium dihydrogen phosphate, ammonium sulfate, and magnesium sulfate. Preferably, the concentration of each component in the seed culture medium is 6-14 g / L yeast powder, 16-24 g / L peptone, 18-26 g / L glucose, 0.5-2.5 g / L potassium dihydrogen phosphate, 1-3 g / L ammonium sulfate, and 0.3-0.7 g / L magnesium sulfate, preferably 8-12 g / L yeast powder, 18-22 g / L peptone, 20-24 g / L glucose, 1-2 g / L potassium dihydrogen phosphate, 1.5-2.5 g / L ammonium sulfate, and 0.4-0.6 g / L magnesium sulfate, and more preferably 10 g / L yeast powder, 20 g / L peptone, 22 g / L glucose, 1.5 g / L potassium dihydrogen phosphate, 2 g / L ammonium sulfate, and 0.5 g / L magnesium sulfate. That is, the secondary seed liquid is the seed liquid of the fermentation tank, and the culture using the secondary seed liquid in the fermentation tank is the adhesion culture of yeast cells on the surface of the solid carrier.

[0039] In some embodiments, when the ethanol content in the fermentation broth is above 5 g / L, the fermentation broth is transferred to a membrane separation module for membrane separation to obtain ethanol. The separated fermentation broth then enters the fermentation broth system again for surface adhesion fermentation. The device is shown in FIG14 . The separation membrane in the membrane separation module is a pervaporation membrane. Preferably, the material of the separation membrane in the membrane separation module is polydimethylsiloxane. Preferably, the area of ​​the separation membrane in the membrane separation module is 75-500 cm 2 , preferably 300-400cm 2 , more preferably 314cm 2 .

[0040] In a second aspect, the present invention discloses a method for continuously producing ethanol by coupling surface adhesion fermentation with membrane separation, wherein brewer's yeast is inoculated into a fermentation system containing a solid carrier to carry out surface adhesion fermentation to produce ethanol, the obtained fermentation liquid is transferred to a membrane separation component for membrane separation to obtain ethanol, and the separated fermentation liquid enters the fermentation liquid system again for surface adhesion fermentation; wherein, after the membrane separation begins, the membrane separation and adhesion fermentation are carried out simultaneously to form a cycle; wherein, the membrane separation can remove the ethanol in the fermentation system, eliminate the inhibitory effect of ethanol on bacterial cells, and at the same time, the ethanol can be separated and collected, thereby simplifying the fermentation process and realizing a continuous process flow.

[0041] Wherein, the brewer's yeast is a recombinant brewer's yeast expressing lectin protein, and the adhesion ability of the yeast cells is enhanced by genetic engineering technology; preferably, the lectin protein is any one or a combination of FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3 and ALS5, preferably FLO5 and / or FLO10.

[0042] The accession numbers of the amino acid sequences of FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3 and ALS5 are NP_009424.1, NP_012081.1, NP_009338.1, NP_013028.1, NP_012284.3, XP_718077.1, XP_710435.2, XP_718 074.2; the registration numbers of the nucleotide sequences of FLO1, FLO5, FLO9, FLO10, and FLO11 are NM_001178230.1, NM_001179342.1, NM_001178205.1, NM_001179892.1, and NM_001179541.3, respectively; the nucleotide sequences of ALS1, ALS3, and ALS5 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0043] Wherein, the expression is integrated expression.

[0044] The genomic integration site for integration expression includes any chromosome of Saccharomyces cerevisiae chromosomes 1-16, preferably, the chromosomes are chromosomes 1, 4, and 16, and more preferably, the insertion site for integration expression is 106a, 416d, or 1622b.

[0045] The integrated expression method includes homologous recombination, CRISPR gene editing technology, etc., preferably CRISPR / Cas9 gene editing technology; the promoter used for expression is a constitutive promoter, preferably any one of TPI, TEF1 and TDH3; the terminator used for expression is CYC1 or ADH1.

[0046] The construction of the recombinant Saccharomyces cerevisiae strain comprises the following steps:

[0047] (1) Replacing the 20-nt sequence in the CRISPR / Cas9 gene editing plasmid with the sequence of the gene integration site to obtain a reconstructed CRISPR / Cas9 gene editing plasmid, such as pCAS-1622b;

[0048] (2) Extracting the genome of Saccharomyces cerevisiae;

[0049] (3) Using the Saccharomyces cerevisiae genome as a template, the upstream homology arm sequence, downstream homology arm sequence, promoter sequence, target base sequence, and terminator sequence were obtained by PCR;

[0050] (4) connecting the gene fragments described in (3) in the order of upstream homologous arm-promoter-target gene-terminator-downstream homologous arm to obtain homologous recombinant fragments;

[0051] (5) The reconstructed CRISPR / Cas9 gene editing plasmid and the homologous recombination fragment described in (1) and (4) are transferred into Saccharomyces cerevisiae to obtain a recombinant Saccharomyces cerevisiae strain.

[0052] In step (1), the 20-nt sequence (1622b) of the gene integration site in chromosome 16 is shown as SEQ ID NO.4.

[0053] In step (2), the brewer's yeast is Saccharomyces cerevisiae CICC1308. In step (3), the promoter used is a constitutive promoter; preferably, the promoter used in the integrated expression is TPI or any one of TEF1 and TDH3; preferably, the terminator used in the integrated expression is CYC1 or ADH1.

[0054] In step (3), the target genes FLO1, FLO5, FLO9, FLO10, and FLO11 were obtained by PCR using the genome of Saccharomyces cerevisiae CICC1308 as a template; the target genes ALS1, ALS3, and ALS5 were synthesized by a biotechnology company.

[0055] In step (5), the recombinant Saccharomyces cerevisiae strain is screened in a YPD medium containing 100-600 mg / L G418 antibiotic. Preferably, the recombinant Saccharomyces cerevisiae strain is screened in a YPD medium containing 500 mg / L G418 antibiotic.

[0056] The solid carrier comprises any one of cotton fiber, glass sheet, polyester fiber, nylon fiber, wood pulp cotton, polylactic acid, activated carbon, polyethylene, polyvinyl alcohol, polyurethane, clay, metal and ceramic, preferably cotton fiber.

[0057] The content of the solid carrier is 1-100 g / L culture medium, preferably 30-50 g / L culture medium, and more preferably 35 g / L culture medium; preferably, the culture medium is a seed culture medium.

[0058] The brewer's yeast seed liquid is inoculated into a culture medium at a volume ratio of 1%-50% to produce ethanol, preferably 5%-15%, and more preferably 10%; preferably, the culture medium is a seed culture medium, preferably a seed culture medium containing a solid carrier.

[0059] The seed culture medium used in the fermentation process includes yeast powder, peptone, glucose, potassium dihydrogen phosphate, ammonium sulfate, and magnesium sulfate; preferably, the concentration of each component in the seed culture medium is 6-14 g / L yeast powder, 16-24 g / L peptone, 18-26 g / L glucose, 0.5-2.5 g / L potassium dihydrogen phosphate, 1-3 g / L ammonium sulfate, and 0.3-0.7 g / L magnesium sulfate, preferably 8-12 g / L yeast powder, 18-22 g / L peptone, 20-24 g / L glucose, 1-2 g / L potassium dihydrogen phosphate, 1.5-2.5 g / L ammonium sulfate, and 0.4-0.6 g / L magnesium sulfate, and more preferably 10 g / L yeast powder, 20 g / L peptone, 22 g / L glucose, 1.5 g / L potassium dihydrogen phosphate, 2 g / L ammonium sulfate, and 0.5 g / L magnesium sulfate.

[0060] Wherein, in the fermentation system, the initial concentration of glucose in the fermentation medium is 50-250 g / L, preferably 150-150 g / L, and more preferably 100 g / L; preferably, the fermentation medium further comprises tryptone, yeast powder, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate, zinc sulfate heptahydrate, and ferrous sulfate heptahydrate; preferably, the concentration of each component in the fermentation medium except glucose is tryptone 2-6 g / L, yeast powder 1-5 g / L, potassium dihydrogen phosphate 1-5 g / L, ammonium sulfate 2-6 g / L, magnesium sulfate 0.3-0.7 g / L, zinc sulfate heptahydrate 0.03- 0.07 g / L, ferrous sulfate heptahydrate 0.03-0.07 g / L, preferably tryptone 3-5 g / L, yeast powder 2-4 g / L, potassium dihydrogen phosphate 2-4 g / L, ammonium sulfate 3-5 g / L, magnesium sulfate 0.4-0.6 g / L, zinc sulfate heptahydrate 0.04-0.06 g / L, ferrous sulfate heptahydrate 0.04-0.06 g / L, more preferably tryptone 4 g / L, yeast powder 3 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 4 g / L, magnesium sulfate 0.5 g / L, zinc sulfate heptahydrate 0.05 g / L, ferrous sulfate heptahydrate 0.05 g / L.

[0061] Wherein, in the fermentation system, the turbidity OD of the fermentation liquid is 600nm The value is 30 or less, preferably 25 or less, or 15 or less, or 10 or less, or 5 or less, or 1 or less, preferably 15 or less.

[0062] Among them, the surface adhesion fermentation refers to the yeast cells being able to adhere to a solid carrier to form a biofilm. The biofilm can improve the yeast cells' tolerance to the environment, maintain the cell's growth ability and long-term biological activity. Unlike cross-linking and embedding methods, surface adhesion fermentation can ensure the activity of yeast cells while not affecting the momentum transfer, heat transfer, and mass transfer in the fermentation system. It can reduce the bacterial cell density in the fermentation broth and reduce the turbidity of the fermentation broth.

[0063] In some embodiments, the surface adhesion fermentation is to add a solid carrier to the seed culture medium or fermentation medium at the initial stage of fermentation for fermentation, and after the fermentation is completed, the fermentation liquid is released and the solid carrier is retained, and fresh fermentation medium is added to continue fermentation; preferably, the initial stage of the fermentation is before the first-level seed liquid is accessed to the second-level seed liquid; preferably, the solid carrier is added to the seed culture medium or fermentation medium at the initial stage of fermentation for fermentation is to access the first-level seed liquid to the second-level seed liquid containing the solid carrier for fermentation.

[0064] In some embodiments, the surface adhesion fermentation is to connect the first-level seed liquid to the second-level seed liquid containing the solid carrier for static culture first, and then for flow circulation culture. After the fermentation is completed, the fermentation liquid is released and the solid carrier is retained, and fresh fermentation medium is added to continue fermentation.

[0065] Wherein, the temperature of the static culture and the flow circulation culture is 25-35°C, preferably 28-32°C, and more preferably 30°C; preferably, the static culture time is 1-7h, preferably 3-5h, and more preferably 4h; preferably, the flow circulation culture rate is 10-50mL / min, preferably 20-40mL / min, and more preferably 30mL / min; preferably, the flow circulation culture time is 4-16h, preferably 8-12h; preferably, after the fresh fermentation medium is supplemented, the fermentation broth circulates to start the next batch of fermentation; preferably, the circulation rate of the fermentation broth is 10-50mL / min, preferably 20-40mL / min, and more preferably 30mL / min.

[0066] The released fermentation liquid is fermentation liquid that accounts for more than 95% v / v of the total fermentation liquid, preferably all of the fermentation liquid; preferably, the volume of the fresh fermentation medium added is the same as the volume of the released fermentation liquid.

[0067] Wherein, the fermentation temperature is 25-37°C, preferably 28-37°C, and more preferably 35°C.

[0068] Wherein, when the ethanol content in the fermentation broth is 5 g / L or more, the fermentation broth is transferred to the membrane separation component; preferably, when the ethanol content in the fermentation broth is 5 g / L, or 10 g / L, or 15 g / L, or 20 g / L, or 25 g / L, or 30 g / L, or 35 g / L, or 40 g / L, or 45 g / L, or 50 g / L, or 55 g / L, or 60 g / L, or 65 g / L, or 70 g / L, or 75 g / L, or 80 g / L L, or 85g / L, or 90g / L, or 95g / L, or 100g / L, the fermentation broth is transferred to the membrane separation component; further preferably, when the ethanol content in the fermentation broth is 10g / L, or 15g / L, or 20g / L, or 25g / L, or 30g / L, or 35g / L, or 40g / L, the fermentation broth is transferred to the membrane separation component; even further preferably, when the ethanol content in the fermentation broth is 20g / L, the fermentation broth is transferred to the membrane separation component.

[0069] The fermentation broth is transferred into the membrane separation component at a flow rate of 5-1000 mL / min, preferably 300-500 mL / min, and more preferably 450 mL / min.

[0070] After the fermentation liquid is transferred to the membrane separation module, the concentration of glucose in the fermentation system is maintained at 5-100 g / L, preferably 10-90 g / L, and more preferably 20-80 g / L.

[0071] The separation membrane in the membrane separation assembly is a pervaporation membrane; preferably, the separation membrane in the membrane separation assembly is made of polydimethylsiloxane; preferably, the area of ​​the separation membrane in the membrane separation assembly is 75-500 cm 2 , preferably 300-400cm 2 , more preferably 314cm 2 .

[0072] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0073] (1) The present invention provides a recombinant Saccharomyces cerevisiae strain, which enhances the ethanol production efficiency of Saccharomyces cerevisiae and improves its adhesion and film-forming ability by 0.5-5 times.

[0074] (2) The recombinant Saccharomyces cerevisiae strain constructed by the present invention can promote the adhesion and growth of yeast cells on solid media in the continuous production of ethanol by surface adhesion by improving the ability of yeast cells to adhere to and form a film, thereby realizing a continuous production process. It can also increase the population growth density of cells, enhance the tolerance of cells and resistance to adverse production environments, and improve the reaction performance of yeast cells and the production efficiency of ethanol.

[0075] (3) The method for continuous ethanol production by coupling surface-adhesive fermentation with membrane separation provided by the present invention can reduce fermentation broth turbidity, minimize separation membrane contamination, improve membrane separation efficiency, and reduce the production of fermentation metabolic byproducts such as glycerol and succinic acid. The recombinant strain provided by the present invention has improved adhesion ability, resulting in a lower viscosity of the resulting fermentation broth, which facilitates membrane reaction separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0077] Figure 1 is a plasmid map of the CRISPR / Cas9 gene editing plasmid pCAS-1622b.

[0078] Figure 2 is a plasmid map of the CRISPR / Cas9 gene editing plasmid pCAS-106a.

[0079] Figure 3 is a plasmid map of the CRISPR / Cas9 gene editing plasmid pCAS-416d.

[0080] FIG4 shows the absorbance at 570 nm of the biofilms formed by the recombinant strains constructed by integrating and expressing a single gene into the genome of Saccharomyces cerevisiae BY4742, after staining with crystal violet, which can characterize the biofilm-forming ability of the recombinant strains.

[0081] FIG5 shows the absorbance at 570 nm of the biofilms formed by the recombinant strains constructed by integrating multiple genes into the genome of Saccharomyces cerevisiae BY4742 after staining with crystal violet, which can characterize the biofilm-forming ability of the recombinant strains.

[0082] FIG6 shows the absorbance at 570 nm of the biofilms formed by the recombinant strains constructed by integrating a single gene into the genome of Saccharomyces cerevisiae W303-1A after staining with crystal violet, which can characterize the biofilm-forming ability of the recombinant strains.

[0083] FIG7 shows the absorbance at 570 nm of the biofilms formed by the recombinant strains constructed by integrating multiple genes into the genome of Saccharomyces cerevisiae W303-1A after staining with crystal violet, which can characterize the biofilm-forming ability of the recombinant strains.

[0084] FIG8 shows the absorbance at 570 nm of the biofilms formed by the recombinant strains constructed by integrating and expressing a single gene into the genome of Saccharomyces cerevisiae CICC1308 after staining with crystal violet, which can characterize the biofilm-forming ability of the recombinant strains.

[0085] FIG9 shows the absorbance at 570 nm of the biofilms formed by the recombinant strains constructed by integrating multiple genes into the genome of Saccharomyces cerevisiae CICC1308. The biofilms were stained with crystal violet, which can characterize the biofilm-forming ability of the recombinant strains.

[0086] FIG10 shows the phenomenon of yeast cells aggregating with each other observed under a microscope in each recombinant strain constructed in Saccharomyces cerevisiae BY4742.

[0087] FIG11 shows the phenomenon of yeast cells aggregating with each other observed under a microscope in each recombinant strain constructed in Saccharomyces cerevisiae W303-1A.

[0088] FIG12 shows the phenomenon of yeast cells aggregating with each other observed under a microscope in each recombinant strain constructed in Saccharomyces cerevisiae CICC1308.

[0089] FIG13 shows the bacterial cells adhered to the cotton fiber carrier using a scanning electron microscope (SEM) after the surface adhesion continuous fermentation of each recombinant strain constructed in Saccharomyces cerevisiae CICC1308 was completed.

[0090] FIG14 is a diagram of the experimental setup for coupling surface adhesion fermentation and membrane separation.

[0091] Figure 15 shows the mass of ethanol separated during the membrane reaction separation process.

[0092] Figure 16 shows the changes in glycerol concentration in the 2-L reactor during membrane reaction separation.

[0093] FIG17 shows the change of succinic acid concentration in the 2-L reactor during the membrane reaction separation process.

[0094] FIG18 is a diagram of the experimental setup for coupling surface adhesion fermentation and membrane separation.

[0095] Figure 19 shows the changes in glucose concentration in the 2-L reactor during membrane reaction separation.

[0096] Figure 20 shows the changes in ethanol concentration in the 2-L reactor during membrane reaction separation.

[0097] Figure 21 shows the mass of ethanol separated during the membrane reaction separation process.

[0098] Figure 22 shows the changes in membrane flux during membrane reaction separation.

[0099] Figure 23 shows the somatic cell concentration (OD) in the fermentation broth of the 2-L reactor during membrane reaction separation. 600 value).

[0100] Figure 24 shows the changes in pH value in the 2-L reactor during membrane reaction separation.

[0101] Figure 25 shows the changes in glycerol concentration in the 2-L reactor during membrane reaction separation.

[0102] FIG26 shows the change in succinic acid concentration in the 2-L reactor during membrane reaction separation.

[0103] FIG27 shows the bacterial cells remaining on the surface of the separation membrane after the membrane reaction separation is completed.

[0104] FIG28 shows the biofilm formed on the surface of the solid carrier after the membrane reaction separation is completed. DETAILED DESCRIPTION

[0105] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0106] The culture medium components described in the following examples are shown in Tables 1, 2, 3, and 4, wherein each culture medium was sterilized at 115°C and 0.12 MPa for 20 min and then stored at 4°C for later use;

[0107] Table 1. Liquid LB medium components (solvent is water)

[0108] Table 2. Liquid YPD medium components (solvent is water)

[0109] Table 3. Fermentation medium components (solvent is water) - for enhancing ethanol production by Saccharomyces cerevisiae

[0110] Table 4. Fermentation medium components (solvent is water) - for continuous ethanol production coupled with surface-adhesive fermentation and membrane separation

[0111] The solid plate culture medium described in the following examples was prepared as follows: 20 g / L agar powder was added to the corresponding liquid culture medium and sterilized at 115°C, 0.12 MPa for 20 min. When the culture medium cooled to 60°C, 20 mL of the culture medium was poured into a sterile plate culture dish in a clean bench (antibiotics may be added as needed). After the culture medium solidified, it was stored at 4°C for later use.

[0112] The seed culture medium in the following examples is composed of 10 g / L yeast powder, 20 g / L peptone, 22 g / L glucose, 1.5 g / L potassium dihydrogen phosphate, 2 g / L ammonium sulfate, and 0.5 g / L magnesium sulfate, and the pH is adjusted to 5.5.

[0113] The one-step cloning reaction system described in the following examples is shown in Table 5:

[0114] Table 5. One-step cloning reaction system

[0115] The residual sugar concentrations described in the following examples were determined using an SBA-biosensor analyzer. The specific determination method was as follows: (1) The sample was centrifuged at 10,000 rpm for 2 minutes, 1 mL of the supernatant was collected, and the sample was diluted 100-fold using pure water; (2) The SBA-biosensor analyzer was calibrated using the standard solution, and then 10 μL of the sample was collected for detection.

[0116] The ethanol yields described in the following examples were detected by gas chromatography-mass spectrometry, and the specific detection method is as follows:

[0117] (1) The sample was centrifuged at 10,000 rpm for 2 min. 1 mL of the supernatant was taken and diluted 10-fold with pure water. The sample was then filtered through a 0.22 μm filter membrane for later use.

[0118] (2) The ethanol content in the fermentation broth was detected and analyzed using a high-performance gas chromatograph (7890A GC-system, Agilent Technologies, USA). The gas chromatography conditions were as follows: HP-INNOWax capillary column, PEG-20M, 60m×0.25mm×0.5μm; the column temperature was programmed: initially 70°C, maintained for 0.5min, and then increased to 20°C·min -1 The temperature was raised to 190°C at a rate of 1.0 mL / min and maintained for 4 min. -1 , tail blow flow rate 29mL·min -1 , hydrogen flow rate 30 mL·min -1 , air flow rate 300 mL·min -1 The front inlet temperature was 180°C, the FID temperature was 220°C, the injection volume was 1.0 μL, and the split ratio was 10:1.

[0119] In the following examples, glycerol and succinic acid were detected using a liquid high performance chromatography analyzer, and the specific detection method is as follows:

[0120] (1) The sample was centrifuged at 10,000 rpm for 2 min. 1 mL of the supernatant was taken and diluted 10-fold with pure water. The sample was filtered through a 0.22 μm filter membrane and then used for later use.

[0121] (2) The contents of glycerol and succinic acid in the fermentation broth were detected and analyzed using a high performance liquid chromatograph (Agilent 1260 Infinity II, Agilent Technologies, USA). The chromatographic column used was an organic acid analysis column (BIO-RAD Aminex HPX-87H, 300 mm × 7.8 mm). A differential detector was used, the mobile phase was 5 mM sulfuric acid, the flow rate was 0.6 mL / min, and the column oven was set to 60°C.

[0122] The strains constructed in the present invention include BY474-FLO1, BY474-FLO5, BY474-FLO9, BY474-FLO10, BY474-FLO11, BY474-ALS1, BY474-ALS3, BY474-ALS5, BY474-FLO9-FLO11, BY474-FLO11-FLO11, BY474-FLO10-ALS3, BY474-FLO11-ALS3, BY474-ALS3-ALS5, BY474-FLO9-FLO10-FLO11, BY474-FLO10-FLO11-ALS3, W303-1 A-FLO1, W303-1A-FLO5, W303-1A-FLO9, W303-1A-FLO10, W303-1A-FLO11, W303-1A-ALS1, W303-1A-ALS3, W303-1A-ALS5, W303-1A-FLO9-FLO11, W303-1A-FLO5-FLO11, W303-1A-FLO10-ALS3, W303-1A-FLO11-ALS3, W303-1A-ALS3-ALS5, W303-1A-FLO5-FLO10-FLO11, W303-1A-FLO1-FLO9-AL S3, 1308-FLO1, 1308-FLO5, 1308-FLO9, 1308-FLO10, 1308-FLO11, 1308-ALS1, 1308-ALS3, 1308-ALS5, 1308-FLO9-FLO10, 1308-FLO1-FLO10, 1308-FLO5-FLO11, 1308-FLO11-ALS3, 1308-ALS3-ALS5, 1308-FLO5-FLO10-FLO11, 1308-FLO10-ALS1-ALS3. For specific construction methods, please refer to the following Examples 1-7. Among them, the preferred plasmid for single-copy integrated expression is the pCAS-1622b plasmid, the preferred plasmid for double-copy or two-gene strains is pCAS-1622b and pCAS-106a, and the preferred plasmid for three genes is pCAS-1622b, pCAS-106a, and pCAS-416d. Among them, the promoter used at the 1622b site during the integrated expression process is TPI, and the terminator used is CYC1; the promoter used at the 106a site is TEF1, and the terminator used is CYC1; the promoter used at the 416d site is TDH3, and the terminator used is ADH1.

[0123] Example 1. Reconstruction of CRISPR / Cas9 gene editing plasmid pCAS-1622b (Figure 1)

[0124] (1) In a sterile clean bench, 10 μL of E. coli DH5α glycerol culture containing the pCAS plasmid was inoculated into 5 mL of liquid LB medium and cultured overnight at 37°C and 200 rpm. The pCAS plasmid was extracted and digested with SnaBⅠ and BglⅡ restriction enzymes to obtain a linearized vector fragment.

[0125] (2) Using primers pCAS-F, pCAS-1622b-R and pCAS-1622b-F, pCAS-R, respectively, and the pCAS plasmid as a template, two gene fragments were obtained by PCR; then, using primers pCAS-F and pCAS-R, the two gene fragments obtained above were used as templates to obtain the target gene fragment by overlap PCR;

[0126] (3) The linearized vector fragment described in (1) and the target gene fragment described in (2) are connected by one-step cloning. The connection system is shown in Table 5 to obtain a connection solution.

[0127] (4) Take 10 μL of the ligation solution described in (3) and add it to E. coli DH5α competent cells, and place it on ice for 30 minutes; then incubate it at 42°C for 90 seconds and place it on ice for 3-5 minutes; then add 800 μL of fresh LB culture medium in a sterile environment and culture it at 37°C and 200 rpm for 45 minutes; then take 200 μL of culture solution and spread it on LB solid culture medium containing Amp antibiotics, and culture it at 30°C for 12 hours; finally, pick a single colony point, inoculate it into fresh LB liquid culture medium and culture it at 37°C and 200 rpm for 12 hours, collect the bacterial liquid and extract the plasmid, and send it to a biotechnology company for sequencing.

[0128] Table 6. Primer sequences used in this example

[0129] Example 2. Reconstruction of CRISPR / Cas9 gene editing plasmid pCAS-106a (Figure 2)

[0130] (1) In a sterile clean bench, 10 μL of E. coli DH5α glycerol culture containing the pCAS plasmid was inoculated into 5 mL of liquid LB medium and cultured overnight at 37°C and 200 rpm. The pCAS plasmid was extracted and digested with SnaBⅠ and BglⅡ restriction enzymes to obtain a linearized vector fragment.

[0131] (2) Using primers pCAS-F, pCAS-106b-R and pCAS-106a-F, pCAS-R, respectively, and the pCAS plasmid as a template, two gene fragments were obtained by PCR; then, using primers pCAS-F and pCAS-R, the two gene fragments obtained above were used as templates to obtain the target gene fragment by overlap PCR;

[0132] (3) The linearized vector fragment described in (1) and the target gene fragment described in (2) are connected by one-step cloning. The connection system is shown in Table 5 to obtain a connection solution.

[0133] (4) Take 10 μL of the ligation solution described in (3) and add it to E. coli DH5α competent cells, and place it on ice for 30 minutes; then incubate it at 42°C for 90 seconds and place it on ice for 3-5 minutes; then add 800 μL of fresh LB culture medium in a sterile environment and culture it at 37°C and 200 rpm for 45 minutes; then take 200 μL of culture solution and spread it on LB solid culture medium containing Amp antibiotics, and culture it at 30°C for 12 hours; finally, pick a single colony point, inoculate it into fresh LB liquid culture medium and culture it at 37°C and 200 rpm for 12 hours, collect the bacterial liquid and extract the plasmid, and send it to a biotechnology company for sequencing.

[0134] Table 7. Primer sequences used in this example

[0135] Example 3. Reconstruction of CRISPR / Cas9 gene editing plasmid pCAS-416d (Figure 3)

[0136] (1) In a sterile clean bench, 10 μL of E. coli DH5α glycerol culture containing the pCAS plasmid was inoculated into 5 mL of liquid LB medium and cultured overnight at 37°C and 200 rpm. The pCAS plasmid was extracted and digested with SnaBⅠ and BglⅡ restriction enzymes to obtain a linearized vector fragment.

[0137] (2) Using primers pCAS-F, pCAS-416d-R and pCAS-416d-F, pCAS-R, respectively, and the pCAS plasmid as a template, two gene fragments were obtained by PCR; then, using primers pCAS-F and pCAS-R, the two gene fragments obtained above were used as templates to obtain the target gene fragment by Overlap PCR;

[0138] (3) The linearized vector fragment described in (1) and the target gene fragment described in (2) are connected by one-step cloning. The connection system is shown in Table 5 to obtain a connection solution.

[0139] (4) Take 10 μL of the ligation solution described in (3) and add it to E. coli DH5α competent cells, and place it on ice for 30 minutes; then incubate it at 42°C for 90 seconds and place it on ice for 3-5 minutes; then add 800 μL of fresh LB culture medium in a sterile environment and culture it at 37°C and 200 rpm for 45 minutes; then take 200 μL of culture solution and spread it on LB solid culture medium containing Amp antibiotics, and culture it at 30°C for 12 hours; finally, pick a single colony point, inoculate it into fresh LB liquid culture medium and culture it at 37°C and 200 rpm for 12 hours, collect the bacterial liquid and extract the plasmid, and send it to a biotechnology company for sequencing.

[0140] Table 8. Primer sequences used in this example

[0141] Example 4. Construction of homologous recombination fragments

[0142] 1. Single copy integrated expression, i.e. construction of 1622b site homologous recombination fragment

[0143] The homologous recombination fragment expressing FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3, and ALS5 genes was constructed on chromosome 16 (site 1622b) of Saccharomyces cerevisiae. The promoter used for constructing the homologous recombination fragment at this site was TPI, and the terminator used was CYC1. The specific construction method is as follows:

[0144] (1) Using the genome of Saccharomyces cerevisiae CICC1308, BY4742, or W303-1A as a template, and using primers 1622b-up-F, 1622b-up-R, and 1622b-down-F, 1622b-down-R, respectively, upstream and downstream homology arm fragments were obtained by PCR;

[0145] (2) Using the genome of Saccharomyces cerevisiae CICC1308, BY4742 or W303-1A as a template and 1622b-TPI-F and 1622b-TPI(FLO1)-R as primers, the gene fragment of the TPI-(FLO1) promoter was obtained by PCR; using 1622b-TPI-F and 1622b-TPI(FLO5)-R as primers, the gene fragment of the TPI-(FLO5) promoter was obtained by PCR; using 1622b-TPI-F and 1622b-TPI(FLO9)-R as primers, the gene fragment of the TPI-(FLO9) promoter was obtained by PCR; using 1622b-TPI-F and 1622b-TPI(FLO10)-R as primers, the gene fragment of the TPI-(FLO11) promoter was obtained by PCR. The gene fragment of TPI-(FLO10) promoter was obtained by PCR using 1622b-TPI-F and 1622b-TPI(FLO11)-R as primers; the gene fragment of TPI-(ALS1) promoter was obtained by PCR using 1622b-TPI-F and 1622b-TPI(ALS1)-R as primers; the gene fragment of TPI-(ALS3) promoter was obtained by PCR using 1622b-TPI-F and 1622b-TPI(ALS3)-R as primers; the gene fragment of TPI-(ALS5) promoter was obtained by PCR using 1622b-TPI-F and 1622b-TPI(ALS5)-R as primers;

[0146] (3) Using the pYES2 / CT-α-factor plasmid as a template and 1622b-CYC1t(FLO1)-F and 1622b-CYC1t-R as primers, the gene fragment of CYC1t-(FLO1) terminator was obtained by PCR. Using 1622b-CYC1t(FLO5)-F and 1622b-CYC1t-R as primers, the gene fragment of CYC1t-(FLO5) terminator was obtained by PCR. Using 1622b-CYC1t(FLO9)-F and 1622b-CYC1t-R as primers, the gene fragment of CYC1t-(FLO9) terminator was obtained by PCR. Using 1622b-CYC1t(FLO10)-F and 1622b-CYC1t-R as primers, the gene fragment of CYC1t-(FLO10) terminator was obtained by PCR. LO10) terminator gene fragment; using 1622b-CYC1t(FLO11)-F and 1622b-CYC1t-R as primers, the CYC1t-(FLO11) terminator gene fragment was obtained by PCR; using 1622b-CYC1t(ALS1)-F and 1622b-CYC1t-R as primers, the CYC1t-(ALS1) terminator gene fragment was obtained by PCR; using 1622b-CYC1t(ALS3)-F and 1622b-CYC1t-R as primers, the CYC1t-(ALS3) terminator gene fragment was obtained by PCR; using 1622b-CYC1t(ALS5)-F and 1622b-CYC1t-R as primers, the CYC1t-(ALS5) terminator gene fragment was obtained by PCR;

[0147] (4) Using the genome of Saccharomyces cerevisiae CICC1308, BY4742 or W303-1A as a template, TPI-FLO1-F and CYC1t-FLO1-R were used as primers to obtain the FLO1 gene fragment by PCR; TPI-FLO5-F and CYC1t-FLO5-R were used as primers to obtain the FLO5 gene fragment by PCR; TPI-FLO9-F and CYC1t-FLO1-R were used as primers to obtain the FLO9 gene fragment by PCR; TPI-FLO10-F and CYC1t-FLO10-R were used as primers, The FLO10 gene fragment was obtained by PCR; the FLO11 gene fragment was obtained by PCR using TPI-FLO11-F and CYC1t-FLO11-R as primers respectively; the ALS1 gene fragment was obtained by PCR using TPI-ALS1-F and CYC1t-ALS1-R as primers respectively; the ALS3 gene fragment was obtained by PCR using TPI-ALS3-F and CYC1t-ALS3-R as primers respectively; the ALS5 gene fragment was obtained by PCR using TPI-ALS5-F and CYC1t-ALS5-R as primers respectively;

[0148] (5) Using the gene fragments obtained in (1), (2), (3), and (4) above as templates, and 1622b-up-F and 1622b-down-R as templates, the homologous recombinant fragments ultimately used for the construction of the recombinant strain were obtained by Overlap PCR.

[0149] 2. Double copy or two gene combination expression, that is, construction of homologous recombination fragments at sites 1622b and 106a. The double copy or two gene combination expression described in this example is further constructed on the basis of the recombinant strain constructed by single gene integration expression (the CRISPR / Cas9 gene editing plasmid has been eliminated, and the specific elimination method is as described in Example 7).

[0150] The homologous recombination fragment expressing FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3, and ALS5 genes was constructed in chromosome 1 (site 106a) of Saccharomyces cerevisiae. The promoter used for constructing the homologous recombination fragment at this site was TPI, and the terminator used was CYC1. The specific construction method is as follows:

[0151] (1) Using the genome of Saccharomyces cerevisiae CICC1308, BY4742, or W303-1A as a template, and using primers 106a-up-F, 106a-up-R, and 106a-down-F, 106a-down-R, respectively, upstream and downstream homology arm fragments were obtained by PCR;

[0152] (2) Using the homologous recombination fragment used in the constructed 1622b site as a template, 106a-TPI-F as an upstream primer, and 106a-CYC1t-R as a downstream primer, the promoter-target gene-terminator gene fragment used in the 106a site was obtained by PCR.

[0153] (3) Using the gene fragments obtained in (1) and (2) above as templates, and 106a-up-F and 106a-down-R as templates, the homologous recombinant fragments ultimately used for the construction of the recombinant strain were obtained by Overlap PCR.

[0154] 3. Expression of three copies or three genes in combination, i.e., construction of homologous recombination fragments at sites 1622b, 106a, and 416d. The three copies or three gene combinations described in this example are further constructed based on the recombinant strain constructed by the integration and expression of two gene combinations or two copies of genes (the CRISPR / Cas9 gene editing plasmid has been eliminated, and the specific elimination method is as described in Example 7). Construction of a homologous recombination fragment that integrates and expresses the FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3, and ALS5 genes on chromosome 4 (site 416d) of Saccharomyces cerevisiae. The promoter used to construct the homologous recombination fragment is TPI, and the terminator used is CYC1. The specific construction method is as follows:

[0155] (1) Using the genome of Saccharomyces cerevisiae CICC1308, BY4742, or W303-1A as a template, and using primers 416da-up-F, 416d-up-R, and 416d-down-F, 416d-down-R, respectively, upstream and downstream homology arm fragments were obtained by PCR;

[0156] (2) Using the homologous recombination fragment used in the constructed 1622b site as a template, 416d-TPI-F as an upstream primer, and 416d-CYC1t-R as a downstream primer, the promoter-target gene-terminator gene fragment used in the 416d site was obtained by PCR.

[0157] (3) Using the gene fragments obtained in (1) and (2) above as templates, and 416d-up-F and 416d-down-R as templates, the homologous recombinant fragments finally used for the construction of the recombinant strain were obtained by Overlap PCR.

[0158] Table 9. Primer sequences used in this example

[0159] Example 5. Preparation of Saccharomyces cerevisiae competent cells

[0160] (1) Activate the Saccharomyces cerevisiae cells by streaking on a solid YPD plate and incubate at 30°C for about 40 hours until a single colony grows;

[0161] (2) Inoculate the activated single colony of Saccharomyces cerevisiae described in step (1) into 5 mL of liquid YPD medium, culture overnight at 30°C and 220 rpm, and then inoculate 1 mL of the bacterial solution into 50 mL of fresh liquid YPD medium to ensure that the initial inoculation OD 600 =0.2, then cultured at 30°C, 220 rpm until OD 600 =1.3;

[0162] (3) In a clean bench, dispense the bacterial solution described in step (2) into sterilized 50 mL centrifuge tubes, 25 mL per tube, and place on ice for 5 min.

[0163] (4) Centrifuge at 4000 rpm, 4°C for 5 min, collect the cells, and add 25 mL of pre-cooled sterile water to each tube for resuspending; Centrifuge at 4000 rpm, 4°C for 5 min, discard the supernatant, and add 12.5 mL of pre-cooled sterile water to each tube for resuspending;

[0164] (5) Centrifuge at 4000 rpm and 4°C for 5 min, discard the supernatant, add 5 mL of pre-cooled sorbitol for resuspending, centrifuge at 4000 rpm and 4°C for 5 min, and discard the supernatant;

[0165] (6) Add 2.5 mL of pre-chilled sorbitol to resuspend the cells and aliquot into pre-chilled 1.5 mL sterile centrifuge tubes, 80 μL per tube, for later use.

[0166] Example 6. Electroporation and screening of recombinant strains

[0167] (1) Take 200 ng of the constructed CRISPR / Cas9 gene editing plasmid pCAS-1622b or pCAS-106a or pCAS-416d and 1000 ng of the corresponding homologous recombination fragment, mix them evenly with the prepared Saccharomyces cerevisiae competent cells, and transfer them to a 2 mm electroporation cuvette. Place them on ice for 5 min and then electroporate them at 1.5 kV, 25 μF, and 200 Ω.

[0168] (2) Immediately after transformation, add 800 μL of fresh YPD medium to the electroporation cuvette and then recover at 30°C and 220 rpm for 1 h.

[0169] (3) The revived bacterial solution was centrifuged at 4000 rpm for 1 min, the supernatant was removed in a sterile clean bench, and 200 μL of fresh YPD medium was added for suspension;

[0170] (4) Take 200 μL of bacterial suspension and spread it on YPD+G418 solid screening plate, incubate it at 30°C for 48 h, and obvious transformants will grow on the solid plate.

[0171] (5) Pick the transformants from (4), transfer them to fresh YPD+G418 solid screening plates for expansion culture, and culture them statically at 30°C for 24 h;

[0172] (6) A small amount of bacterial sludge was selected and placed in a 20 mM NaOH solution and incubated at 95°C for 10 min as a PCR template. Based on the primer sequences shown in Table 10, 1622b-F, 1622b-R, 106a-F, 106a-R, 416d-F, and 416d-R were used as primers to screen the recombinant strains with successful integration by PCR.

[0173] Table 10. Primer sequences used in this example

[0174] Example 7. Elimination of CRISPR / Cas9 gene editing plasmids

[0175] (1) For all successfully constructed recombinant strains, single colonies were picked and inoculated into 5 mL of YPD liquid medium and cultured at 30°C and 220 rpm for 20 h.

[0176] (2) The culture medium in (1) was transferred to 5 mL of fresh YPD liquid medium at a volume ratio of 10%, and cultured at 30°C and 220 rpm for 20 h;

[0177] (3) After two subcultures, the bacterial solution was diluted 100,000 times, spread onto a YPD solid plate, and cultured at 30°C until a single colony grew;

[0178] (4) Pick the same single colony and transfer it to YPD and YPD+G418 solid plate culture medium respectively and culture them under the same conditions for 20 h;

[0179] (3) If the single colony picked can grow normally on the YPD plate but cannot grow on the YPD+G418 plate, it is considered that the plasmid is successfully eliminated, and the recombinant strain with the correct genotype is further screened by colony PCR.

[0180] Example 8. Characterization of biofilm formation ability by crystal violet staining

[0181] (1) Activate Saccharomyces cerevisiae and its recombinant strains by streaking on solid YPD plates and incubate at 30°C for about 40 hours until a single colony grows;

[0182] (2) Inoculate a single colony of the activated Saccharomyces cerevisiae described in step (1) into 5 mL of liquid YPD medium and culture at 30° C. and 220 rpm for 20 h as a seed solution for later use;

[0183] (3) After the seed liquid culture described in (2) is completed, the OD of each bacterial culture liquid is measured. 600 value, and dilute to OD using sterile water in a sterile environment 600 =1.0;

[0184] (4) 20 μL of bacterial culture fluid and 180 μL of fresh culture medium were added to a 96-well plate and incubated at 30°C for 72 h.

[0185] (5) After the culture is completed, the culture medium in the 96-well plate is gently and slowly poured out, and the 96-well plate is repeatedly washed twice with 200 μL PBS to remove the remaining free cells in the well plate;

[0186] (6) Add 200 μL of 0.1% crystal violet staining solution to the 96-well plate and stain at room temperature for 15 min. After staining, wash the 96-well plate twice with 200 μL of PBS to remove the crystal violet that is not bound to the biofilm.

[0187] (7) Add 200 μL of 33% glacial acetic acid to a 96-well plate, shake it at 150 rpm for 45 min, and measure the absorbance at 570 nm using a multifunctional microplate reader.

[0188] The results, shown in Figures 4-7, show that single-copy integration of FLO and ALS family genes into the model Saccharomyces cerevisiae strains BY4742 and W303-1A was used to quantitatively analyze the biofilm-forming ability of each recombinant strain using crystal violet staining. The results showed that all recombinant strains exhibited significantly enhanced biofilm-forming abilities compared to the original control strains. Recombinant strains constructed with single-copy genes in the BY4742 series exhibited 0.5-2.5-fold increased biofilm-forming abilities compared to the original strains; recombinant strains constructed with single-copy genes in the W303-1A series exhibited 0.8-2.4-fold increased biofilm-forming abilities compared to the original strains; recombinant strains constructed with multiple copies or combinations of genes in BY4742 exhibited 1.0-3.3-fold increased biofilm-forming abilities compared to the original strains; and recombinant strains constructed with multiple copies or combinations of genes in W303-1A exhibited 0.8-2.3-fold increased biofilm-forming abilities compared to the original strains. As shown in Figures 8 and 9, the biofilm-forming ability of the recombinant strains constructed by integrating and expressing the FLO5 and FLO8 genes in the genome of Saccharomyces cerevisiae CICC1308 was 0.4 and 0.6 times higher than that of the original strain, respectively. At the same time, the biofilm-forming ability of the recombinant strains constructed after integrating and expressing the FLO family genes and ALS family genes in the single-copy genome of the industrial strain CICC1308 was 0.2-0.7 times higher than that of the original strain; the biofilm-forming ability of the recombinant strains constructed by multi-copy or multi-gene combinations was 0.4-0.8 times higher than that of the original strain.

[0189] Example 9. Characterization and analysis of the aggregation ability of Saccharomyces cerevisiae cells

[0190] (1) Activate Saccharomyces cerevisiae and its recombinant strains by streaking on solid YPD plates and incubate at 30°C for about 40 hours until a single colony grows;

[0191] (2) Inoculate a single colony of the activated Saccharomyces cerevisiae described in step (1) into 5 mL of liquid YPD medium and culture at 30° C. and 220 rpm for 20 h as a seed solution for later use;

[0192] (3) After the seed liquid culture described in (2) is completed, the OD of each bacterial culture liquid is measured. 600 value, and dilute to OD using sterile water in a sterile environment 600 =1.0;

[0193] (4) Add 200 μL of bacterial culture fluid and 5 mL of culture medium to a 6-well plate containing cell slides, and then incubate at 30°C for 72 h.

[0194] (5) After the culture is completed, gently and slowly remove the culture medium in the culture dish, then add 3 mL of PBS to wash the cell slides twice;

[0195] (6) Add 1 mL of pre-cooled 2% glutaraldehyde cell fixative to the culture dish and fix the cells at room temperature for 10 min. Then remove the cell fixative.

[0196] (4) Wash the cell slides twice with 3 mL of PBS, and then place the cell slides under a microscope to observe the aggregation ability of the yeast cells.

[0197] The results are shown in Figures 10-12. When the cell aggregation ability of each recombinant strain was observed under a microscope, the cells of the original strains Saccharomyces cerevisiae BY4742, W303-1A, and CICC1308 were dispersed and not aggregated, while the cells of the constructed recombinant strains were aggregated into clumps. This experimental result shows that the aggregation ability between cells of the constructed recombinant strains was significantly enhanced.

[0198] Example 10. Activation and seed culture of Saccharomyces cerevisiae strains

[0199] (1) In a sterile laminar flow hood, use a sterile inoculating loop to pick up an appropriate amount of Saccharomyces cerevisiae glycerol culture and streak it onto a YPD solid plate;

[0200] (2) The streaked activated plates were placed in a static culture at 30°C for 40 h;

[0201] (3) In a sterile clean bench, use a sterile inoculating loop to transfer all colonies to 200 mL of liquid YPD medium and culture at 30°C and 220 rpm for 20 h to prepare the seed solution for later use.

[0202] Example 11. Continuous Surface Adhesion Fermentation for Ethanol Production in a 2-L Reactor

[0203] In this example, a 2-L column reactor (hereinafter referred to as a 2-L reactor) was used to carry out a surface adhesion continuous fermentation experiment.

[0204] (1) A 2-L reactor containing 1.6 L of seed culture medium was filled with 35 g / L of cleaned cotton fiber as a solid carrier, and then sterilized at 115 °C;

[0205] (2) The seed solution described in Example 10 was injected into a 2-L reactor at a volume ratio of 10% by peristaltic pump, and the surface adhesion culture was carried out at 30°C for 4 h; then, the circulation rate of the seed solution was adjusted to 30 mL / min, and the culture was carried out at 30°C for 10 h;

[0206] (3) Finally, the seed liquid in the 2-L reactor was completely drained and an equal volume of fresh fermentation medium was added to begin fermentation. During the fermentation process, the temperature was controlled at 35°C and the fermentation liquid circulation rate was 30 mL / min. Samples were taken every 6 h to test ethanol production, glucose content, pH value, and bacterial cell concentration in the fermentation liquid. When the glucose content was lower than 5 g / L, the fermentation batch was considered complete. All the fermentation liquid was drained and an equal volume of fresh fermentation liquid was added to begin the next batch of fermentation. The results are shown in Tables 11 and 12.

[0207] Table 11. Comparison of ethanol production by recombinant bacterial strains constructed by single gene integration expression in a 2-L reactor surface-adherent continuous fermentation system

[0208] Table 12. Comparison of ethanol production by recombinant strains constructed by multi-gene integration expression in a 2-L reactor surface-adherent continuous fermentation system.

[0209] Example 12. SEM Characterization of Yeast Cells Adhering to Solid Supports

[0210] (1) After the ethanol fermentation is completed, 1 cm × 1 cm cotton fibers were taken from the reactor and washed twice in PBS buffer to remove the floating cells adhering to the cotton fiber carrier;

[0211] (2) Place the cotton fiber carrier described in (1) in 3% glutaraldehyde and fix it at 4°C for 2 hours. Aspirate the fixative and rinse it twice with PBS for 10 minutes each time. Then, fix it with 1% osmium acid pre-cooled at 4°C for 1 hour at 4°C and then rinse it twice with PBS for 10 minutes each time.

[0212] (3) Dehydration: Dehydrate the solid support using gradient alcohol (30%, 50%, 70%, 80%, 90%, 95%, 100%), dehydrating twice with each concentration of alcohol, each time for 15 min.

[0213] (4) Run the critical point drying procedure as follows: fill the system with liquid carbon dioxide to replace ethanol with liquid carbon dioxide, and this process lasts for 20-60 minutes; then increase the temperature and pressure to reach the critical point of carbon dioxide, and maintain it for 4 minutes; maintain the temperature and pressure and slowly release carbon dioxide, and after about 30 minutes, remove the sample.

[0214] (5) The sample is placed on a sample table about 10-15 cm away from the evaporation source. The sample is rotated and sprayed with gold (10KV 220s). The spraying should be uniform and observed under a microscope after completion.

[0215] The results are shown in Figure 13. After the ethanol fermentation was completed, the biofilm formed on the solid carrier was observed and analyzed by SEM. The original strain had fewer yeast cells adhering to the surface of the solid carrier, while the recombinant strain with modified biofilm-forming ability had a large number of yeast cells adhering to the surface of the solid carrier. This experimental result shows that the recombinant strain has better adhesion and biofilm-forming ability than the original strain.

[0216] Example 13. Continuous production of ethanol by coupling surface adhesion fermentation with membrane separation

[0217] In this example, a 2-L column reactor (hereinafter referred to as a 2-L reactor) was used for surface adhesion fermentation. The specific experimental procedures are as follows:

[0218] (1) A 2-L reactor containing 1.6 L of seed culture medium was filled with 35 g / L of cleaned cotton fiber as a solid carrier, and then sterilized at 115 °C;

[0219] (2) The seed solution described in Example 10 was injected into a 2-L reactor at a volume ratio of 10% by peristaltic pump, and surface adhesion culture was carried out at 30°C for 4 h. The circulation rate of the seed solution was then adjusted to 30 mL / min, and the cells were cultured at 30°C for 10 h.

[0220] (3) All the seed liquid in the 2-L reactor was released and an equal volume of fresh fermentation medium was added to start fermentation culture. During the fermentation culture, the temperature was controlled at 35°C and the circulation rate of the fermentation liquid was 30 mL / min.

[0221] (4) When the ethanol concentration in the 2-L reactor reaches 20 g / L, the discharge port of the 2-L reactor is connected to the feed port of the membrane separation assembly, and the discharge port of the membrane assembly is connected to the feed port of the 2-L reactor, thereby coupling surface adhesion fermentation with membrane separation. The specific experimental setup is shown in FIG14 ; the membrane separation is pervaporation membrane separation, and the separation membrane is made of polydimethylsiloxane;

[0222] (5) After the membrane reaction separation coupling started, the flow rate of the fermentation liquid on the separation membrane surface was set to 450 mL / min, and the area of ​​the separation membrane was 314 cm 2 ;

[0223] (6) After the membrane reaction separation begins, a glucose solution with a concentration of 500 g / L is added to the 2-L reactor every 8 hours to ensure that the glucose concentration in the 2-L reactor is maintained at 0-80 g / L as much as possible; samples are taken every 4 hours to detect the glucose, ethanol, glycerol, and succinic acid in the reactor, as well as the concentration and volume of the separated ethanol.

[0224] The mass of ethanol isolated in the 2-L reactor is shown in Figure 15. The recombinant strain modified for biofilm formation isolated a higher amount of ethanol than the wild-type strain. The glycerol and succinic acid concentrations in the 2-L reactor are shown in Figures 16 and 17, respectively. These experimental results demonstrate that the recombinant strain significantly reduced the production of glycerol and succinic acid as byproducts during the fermentation-membrane separation process. This also suggests that the recombinant strain modified for biofilm formation is beneficial in reducing byproduct production during the fermentation-membrane separation process.

[0225] Example 14. Continuous production of ethanol by coupling surface adhesion fermentation with membrane separation

[0226] In this example, a 2-L column reactor (hereinafter referred to as a 2-L reactor) was used for surface adhesion fermentation. The specific experimental procedures are as follows:

[0227] (1) A 2-L reactor containing 1.6 L of seed culture medium was filled with 35 g / L of cleaned cotton fiber as a solid carrier, and then sterilized at 115 °C;

[0228] (2) The seed solution described in Example 10 was injected into a 2-L reactor at a volume ratio of 10% by peristaltic pump, and surface adhesion culture was carried out at 30°C for 4 h. The circulation rate of the seed solution was then adjusted to 30 mL / min, and the cells were cultured at 30°C for 10 h.

[0229] (3) All the seed liquid in the 2-L reactor was released and an equal volume of fresh fermentation medium was added to start fermentation culture. During the fermentation culture, the temperature was controlled at 35°C and the circulation rate of the fermentation liquid was 30 mL / min.

[0230] (4) When the ethanol concentration in the 2-L reactor reaches 20 g / L, the discharge port of the 2-L reactor is connected to the feed port of the membrane separation assembly, and the discharge port of the membrane assembly is connected to the feed port of the 2-L reactor, thereby coupling surface adhesion fermentation with membrane separation. The specific experimental setup is shown in FIG18 ; the membrane separation is pervaporation membrane separation, and the separation membrane is made of polydimethylsiloxane;

[0231] (5) After the membrane reaction separation coupling started, the flow rate of the fermentation liquid on the separation membrane surface was set to 450 mL / min, and the area of ​​the separation membrane was 314 cm 2 ;

[0232] (6) After the membrane reaction separation begins, a glucose solution with a concentration of 500 g / L is added to the 2-L reactor every 8 hours to ensure that the glucose concentration in the 2-L reactor is maintained at 0-80 g / L as much as possible; samples are taken every 4 hours to detect the glucose, ethanol, glycerol, and succinic acid in the reactor, as well as the concentration and volume of the separated ethanol.

[0233] The glucose and ethanol concentrations within the 2-L reactor are shown in Figures 19 and 20, respectively, and the mass of the separated ethanol is shown in Figure 21. The recombinant strain modified for biofilm formation separated a higher amount of ethanol than the wild-type strain. As shown in Figures 22, 23, 27, and 28, the membrane separation flux of the recombinant strain modified for biofilm formation was higher than that of the wild-type strain, and the OD value of the recombinant strain within the 2-L reactor was lower than that of the wild-type strain. Furthermore, after the membrane reaction and separation were completed, only a small number of bacterial cells of the recombinant strain remained on the surface of the separation membrane, while a large number of bacterial cells of the wild-type strain remained on the surface of the separation membrane. These experimental results indicate that the recombinant strain modified for biofilm formation exhibited superior adhesion, significantly reduced the turbidity of the fermentation broth, mitigated membrane contamination by bacterial cells in the fermentation broth, increased membrane separation flux, and enhanced ethanol separation efficiency.

[0234] The pH value in the 2-L reactor is shown in Figure 24, and the concentrations of glycerol and succinic acid in the 2-L reactor are shown in Figures 25 and 26, respectively. The above experimental results show that the production of by-products glycerol and succinic acid in the recombinant strain was significantly reduced during the fermentation-membrane separation process, and thus the pH value of the fermentation broth was lower than that of the wild strain; this experimental result also shows that the recombinant strain modified with biofilm-forming ability is beneficial to reducing the production of by-products during the fermentation-membrane separation process.

[0235] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for enhancing the efficiency of ethanol production by brewer's yeast, characterized in that: The recombinant Saccharomyces cerevisiae is obtained by integrating and expressing the lectin protein into the genome of Saccharomyces cerevisiae. The recombinant Saccharomyces cerevisiae produces ethanol in a fermentation system containing a solid carrier.

2. A method for continuous production of ethanol by coupling surface adhesion fermentation with membrane separation, characterized in that: Saccharomyces cerevisiae is inoculated into a fermentation system containing a solid carrier to carry out surface adhesion fermentation to produce ethanol, the obtained fermentation liquid is transferred to a membrane separation component for membrane separation to obtain ethanol, and the separated fermentation liquid enters the fermentation liquid system again for surface adhesion fermentation; the Saccharomyces cerevisiae is a recombinant Saccharomyces cerevisiae that integrates and expresses lectin protein.

3. The method according to claim 1 or 2, characterized in that: The lectin protein includes any one or a combination of FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3 and ALS5.

4. The method according to claim 1 or 2, characterized in that: The registration numbers of the amino acid sequences of FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3 and ALS5 are NP_009424.1, NP_012081.1, NP_009338.1, NP_013028.1, NP_012284.3, XP_718077.1, XP_710435.2, XP_718074.3, respectively. 2; Preferably, the registration numbers of the nucleotide sequences of FLO1, FLO5, FLO9, FLO10 and FLO11 are NM_001178230.1, NM_001179342.1, NM_001178205.1, NM_001179892.1 and NM_001179541.3, respectively; the nucleotide sequences of ALS1, ALS3 and ALS5 are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

5. The method according to claim 1, characterized in that The brewer's yeast is any one of Saccharomyces cerevisiae S288C and Saccharomyces cerevisiae CICC1308; preferably, the brewer's yeast is any one of Saccharomyces cerevisiae BY4742, Saccharomyces cerevisiae W303-1A and Saccharomyces cerevisiae CICC1308.

6. The method for enhancing the ethanol production efficiency of brewer's yeast according to claim 1, characterized in that: The construction method of the recombinant Saccharomyces cerevisiae is to integrate and express any one or a combination of several genes of FLO1, FLO5, FLO9, FLO10, FLO11, ALS1, ALS3 and ALS5 lectin protein genes into any chromosome of Saccharomyces cerevisiae chromosomes 1-16; preferably, the integration and expression is into Saccharomyces cerevisiae chromosomes 1, 4, or 16; further preferably, the integration and expression is into Saccharomyces cerevisiae chromosomes 1, 4, or 16, and the insertion site of the integration and expression is 106a, 416d, or 1622b.

7. The method according to claim 1 or 2, characterized in that: The promoter used in the integrated expression is a constitutive promoter; preferably, the promoter used in the integrated expression is any one of TPI, TEF1 and TDH3; preferably, the terminator used in the integrated expression is CYC1 or ADH1.

8. The method for enhancing the ethanol production efficiency of brewer's yeast according to claim 1, characterized in that: The seed liquid of the recombinant Saccharomyces cerevisiae is inoculated into the culture medium at a volume ratio of 1%-50% to produce ethanol, preferably 5%-15%, and more preferably 10%.

9. The method according to claim 1 or 2, characterized in that: The solid carrier is any one of glass sheet, cotton fiber, polyester fiber, nylon fiber, wood pulp cotton, polylactic acid, activated carbon, polyethylene, polyvinyl alcohol, polyurethane, clay, metal and ceramic, preferably cotton fiber; preferably, the dosage of the solid carrier is 1-100 g / L culture medium, preferably 30-50 g / L culture medium, and further preferably 35 g / L culture medium.

10. The method according to claim 1, characterized in that The content of glucose in the fermentation medium used in the fermentation process is 5-300 g / L, preferably 60-250 g / L, and more preferably 200 g / L; preferably, the fermentation medium also includes tryptone, yeast powder, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate, zinc sulfate heptahydrate, and ferrous sulfate heptahydrate; preferably, the concentration of each component in the fermentation medium except glucose is tryptone 2-6 g / L, yeast powder 1-5 g / L, potassium dihydrogen phosphate 1-5 g / L, ammonium sulfate 2-6 g / L, magnesium sulfate 0.3-0.7 g / L, zinc sulfate heptahydrate 0.03-0. 0.07g / L, ferrous sulfate heptahydrate 0.03-0.07g / L, preferably tryptone 3-5g / L, yeast powder 2-4g / L, potassium dihydrogen phosphate 2-4g / L, ammonium sulfate 3-5g / L, magnesium sulfate 0.4-0.6g / L, zinc sulfate heptahydrate 0.04-0.06g / L, ferrous sulfate heptahydrate 0.04-0.06g / L, more preferably tryptone 4g / L, yeast powder 3g / L, potassium dihydrogen phosphate 3g / L, ammonium sulfate 4g / L, magnesium sulfate 0.5g / L, zinc sulfate heptahydrate 0.05g / L, ferrous sulfate heptahydrate 0.05g / L.

11. The method according to claim 1, characterized in that: The recombinant cerevisiae yeast produces ethanol by adhesion fermentation in a fermentation system containing a solid carrier.

12. The method according to claim 11, characterized in that The adherent fermentation is a surface adherent continuous fermentation.

13. The method according to claim 12, characterized in that The surface adhesion continuous fermentation is to add a solid carrier to the seed culture medium or fermentation culture medium at the initial stage of fermentation for fermentation. After each batch of fermentation is completed, the fermentation liquid is released and the solid carrier is retained, and fresh fermentation culture medium is added to start fermentation culture, and this cycle is repeated until the fermentation is completed.

14. The method according to claim 13, characterized in that The solid carrier is added to the seed culture medium or fermentation culture medium for fermentation at the initial stage of fermentation, and the first-level seed liquid is inoculated into the second-level seed liquid containing the solid carrier for surface adhesion culture.

15. The method according to claim 13, characterized in that The surface adhesion continuous fermentation is to connect the first seed liquid to the second seed liquid containing the solid carrier for static culture first, and then flow circulation culture. After the surface adhesion fermentation is completed, the second seed liquid is released and the solid carrier is retained, and fresh fermentation medium is added to start fermentation culture.

16. The method according to claim 15, characterized in that The temperature of the static culture and flow circulation culture is 25-35°C, preferably 28-32°C, and more preferably 30°C; preferably, the time of the static culture is 1-7h, preferably 3-5h, and more preferably 4h; preferably, the rate of the flow circulation culture is 10-50mL / min, preferably 20-40mL / min, and more preferably 30mL / min; preferably, the time of the flow circulation culture is 4-16h, and more preferably 8-12h.

17. The method according to claim 15, characterized in that After the fresh fermentation medium is added, the fermentation liquid circulates and the fermentation culture begins; preferably, the circulation rate of the fermentation liquid is 10-150 mL / min, preferably 20-40 mL / min, and more preferably 30 mL / min.

18. The method according to any one of claims 13 to 15, characterized in that: When the content of glucose in the fermentation medium in the surface adhesion continuous fermentation is below 1 g / L, the batch fermentation is terminated.

19. The method according to any one of claims 13 to 115, characterized in that: The fermentation liquid released is fermentation liquid of more than 95% v / v of the total fermentation liquid, preferably all the fermentation liquid; preferably, the volume of the fresh fermentation medium added is the same as the volume of the fermentation liquid released.

20. The method according to claim 1, characterized in that The fermentation temperature is 25-37°C, preferably 28-37°C, and more preferably 35°C.

21. The method according to claim 2, characterized in that In the fermentation system, the turbidity OD of the fermentation liquid 600nm The value is 30 or less, preferably 25 or less, or 15 or less, or 10 or less, or 5 or less, or 1 or less, preferably 15 or less.

22. The method according to claim 2, characterized in that The surface adhesion fermentation is to add a solid carrier to the seed culture medium or the fermentation culture medium at the initial stage of fermentation for fermentation, and after the fermentation is completed, the fermentation liquid is released and the solid carrier is retained, and fresh fermentation culture medium is added to continue fermentation.

23. The method according to claim 22, characterized in that The fermentation liquid released is fermentation liquid of more than 95% v / v of the total fermentation liquid, preferably all the fermentation liquid; preferably, the volume of the fresh fermentation medium added is the same as the volume of the fermentation liquid released.

24. The method according to claim 2 or 22, characterized in that The fermentation temperature is 25-37°C, preferably 28-37°C, and more preferably 35°C.

25. The method according to claim 2, characterized in that When the ethanol content in the fermentation broth is 5 g / L or more, the fermentation broth is transferred to the membrane separation component; preferably, when the ethanol content in the fermentation broth is 5 g / L, or 10 g / L, or 15 g / L, or 20 g / L, or 25 g / L, or 30 g / L, or 35 g / L, or 40 g / L, or 45 g / L, or 50 g / L, or 55 g / L, or 60 g / L, or 65 g / L, or 70 g / L, or 75 g / L, or 80 g / L, Or 85g / L, or 90g / L, or 95g / L, or 100g / L, the fermentation liquid is transferred to the membrane separation component; further preferably, when the ethanol content in the fermentation liquid is 10g / L, or 15g / L, or 20g / L, or 25g / L, or 30g / L, or 35g / L, or 40g / L, the fermentation liquid is transferred to the membrane separation component; further preferably, when the ethanol content in the fermentation liquid is 20g / L, the fermentation liquid is transferred to the membrane separation component.

26. The method according to claim 2, characterized in that The fermentation broth is transferred into the membrane separation component at a flow rate of 5-1000 mL / min, preferably 300-500 mL / min, and more preferably 450 mL / min.

27. The method according to claim 2, characterized in that After the fermentation liquid is transferred to the membrane separation module, the concentration of glucose in the fermentation system is maintained at 0-100 g / L, preferably 0-90 g / L, and more preferably 0-80 g / L.

28. The method according to claim 2, characterized in that The separation membrane in the membrane separation assembly is a pervaporation membrane; preferably, the material of the separation membrane in the membrane separation assembly is polydimethylsiloxane; preferably, the area of ​​the separation membrane in the membrane separation assembly is 75-500cm 2 , preferably 300-400cm 2 , more preferably 314cm 2 .

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