CN115232757B - Saccharomyces cerevisiae strain, fermentation strain, construction method of saccharomyces cerevisiae strain and bioethanol production method - Google Patents

Abstract

The application relates to the technical field of genetic engineering, in particular to a saccharomyces cerevisiae strain, a fermentation strain, a construction method thereof and a bioethanol production method. The Saccharomyces cerevisiae strain is named as CE10, and the preservation number of the Saccharomyces cerevisiae strain is CGMCC NO.24860. Compared with wild Saccharomyces cerevisiae, the CE10 Saccharomyces cerevisiae strain provided by the application can effectively utilize xylose for metabolism, so that the yield of bioethanol is improved; the tolerance to acid inhibitors such as acetic acid can be improved, and the yield of bioethanol can be further improved.

Description

Saccharomyces cerevisiae strain, fermentation strain, construction method of saccharomyces cerevisiae strain and bioethanol production method

Technical Field

The application relates to the technical field of genetic engineering, in particular to a saccharomyces cerevisiae strain, a fermentation strain, a construction method thereof and a bioethanol production method.

Background

Bioethanol is obtained by taking renewable biomass as a raw material and performing a series of bioconversion processing procedures, and is an important source of renewable biofuel. Compared with gasoline, the bioethanol has higher octane number, higher compression ratio and shorter combustion time, and can effectively reduce the detonation rate of an engine; meanwhile, bioethanol has higher evaporation enthalpy, laminar flame speed and vaporization heat; in addition, the bioethanol has high oxygen content, which is beneficial to improving combustion efficiency, has low sulfur content, and is beneficial to reducing the emission of air pollutants such as sulfur dioxide.

Bioethanol is currently mainly obtained from lignocellulose-rich raw materials such as crop residues (crop stalks, bagasse or corncobs, etc.), energy crops (perennial herbs such as miscanthus or switchgrass, etc.), aquatic plants (water hyacinth, etc.), forest materials, municipal solid biomass, etc., which are abundant, renewable and relatively inexpensive.

The production of ethanol from lignocellulosic biomass involves four steps of pretreatment, hydrolysis, fermentation and distillation. Wherein, the pretreatment process is to remove lignin and hemicellulose from lignocellulose matrix, reduce the crystallinity of cellulose, increase the porosity and the surface area of biomass, and is a key step for converting biomass raw materials into fermentable sugar and biofuel. Fermentation is a process of converting soluble sugar in lignocellulose hydrolysate into bioethanol by using microorganisms such as Saccharomyces cerevisiae and the like. The lignocellulose hydrolysate is rich in glucose, xylose and acetic acid, the xylose is used as the second big sugar in the lignocellulose hydrolysate, and the realization of high-efficiency and rapid conversion of the xylose is an economic means for producing bioethanol by utilizing lignocellulose biomass.

However, the lignocellulose biomass can form acid inhibitors such as acetic acid in the pretreatment process, so that the speed and the yield of the wild type saccharomyces cerevisiae in the subsequent fermentation process can be influenced, the wild type saccharomyces cerevisiae can only use glucose for metabolism, xylose can not be used for metabolism, and further the yield of bioethanol produced by the wild type saccharomyces cerevisiae is low, so that the large-scale production of bioethanol is not facilitated.

Disclosure of Invention

The invention aims to provide a saccharomyces cerevisiae strain, a fermentation strain, a construction method thereof and a bioethanol production method, which aim to solve the technical problems of low xylose utilization efficiency and low bioethanol yield of a wild saccharomyces cerevisiae strain.

In a first aspect, the present application provides a saccharomyces cerevisiae strain named CE10 with a collection number of CGMCC No.24860.

Biological material: CE10, class naming: saccharomyces cerevisiae Saccharomyces cerevisiae, deposited in China general microbiological culture Collection center (CGMCC) at a date of 2022, 05 and 09, has an address of: the collection number of the microbiological institute of China is CGMCC No.24860, and the national institute of sciences of China is No.1, no.3, north Chen West Lu, the Korean region of Beijing city.

Compared with wild Saccharomyces cerevisiae, the CE10 Saccharomyces cerevisiae strain provided by the application can effectively utilize xylose for metabolism, and improves the yield of bioethanol; the tolerance of the strain to acid inhibitors such as acetic acid and the like can be improved, and the yield of bioethanol can be further improved.

In a second aspect, the present application provides a method of constructing a fermentation strain comprising integrating a gene of interest into the genome of the Saccharomyces cerevisiae strain provided in the first aspect above. The target gene is selected from MSN2, TIPI, YRB1 or SPII. Wherein, the MSN2 has a sequence shown as SEQ ID NO.1, the TIPI has a sequence shown as SEQ ID NO.2, the YRB1 has a sequence shown as SEQ ID NO.3, and the SPII has a sequence shown as SEQ ID NO. 4.

The target genes MSN2, TIP1, YRB1 or SPI1 are respectively integrated into genomes of the CE10 saccharomyces cerevisiae strain to construct a fermentation strain, xylose can be effectively utilized to metabolize compared with wild saccharomyces cerevisiae, and compared with the CE10 saccharomyces cerevisiae strain, the utilization efficiency of the strain to xylose and the tolerance of the strain to acid inhibitors such as acetic acid can be further effectively improved, and the yield of bioethanol is further improved.

In a third aspect, the present application provides a method for constructing a fermentation strain, comprising deleting the CAT8 gene in the genome of the saccharomyces cerevisiae strain provided in the first aspect, and integrating the target gene into the genome of the saccharomyces cerevisiae strain deleted of the CAT8 gene by using the CAT8 gene in the genome of the saccharomyces cerevisiae strain as an integration site. The target gene is selected from MSN2, TIPI, YRB1 or SPII. Wherein, the MSN2 has a sequence shown as SEQ ID NO.1, the TIPI has a sequence shown as SEQ ID NO.2, the YRB1 has a sequence shown as SEQ ID NO.3, and the SPII has a sequence shown as SEQ ID NO. 4.

Firstly, CAT8 genes in the genome of the saccharomyces cerevisiae strain CE10 are deleted, so that metabolic pathways of the subsequently prepared fermentation strain during fermentation flow to the ethanol fermentation direction more, and the yield of bioethanol is improved. And then taking CAT8 as an integration site, integrating MSN2, TIP1, YRB1 or SPI1 target genes into the genome of the Saccharomyces cerevisiae strain with the CAT8 genes deleted to construct a fermentation strain, wherein the constructed fermentation strain can remarkably improve the utilization rate of the strain to xylose and the tolerance of the strain to acid inhibitors such as acetic acid, and further improve the yield of bioethanol.

In one possible embodiment, the step of integrating the gene of interest into the genome of the saccharomyces cerevisiae strain after deletion of the CAT8 gene comprises: the linear fragment containing the gene of interest is integrated into the genome of the Saccharomyces cerevisiae strain deleted for CAT8 gene and the gene of interest is overexpressed.

The target gene is over-expressed, which is beneficial to constructing stable fermentation strain.

In a fourth aspect, the present application provides a fermentation strain produced by the method of constructing a fermentation strain as provided in the second or third aspect above.

Compared with wild Saccharomyces cerevisiae, the fermentation strain provided by the application can effectively utilize xylose for metabolism; compared with the CE10 saccharomyces cerevisiae strain, the utilization efficiency of the strain to xylose and the tolerance of the strain to acid inhibitors such as acetic acid can be further effectively improved, and the yield of bioethanol is further improved.

In a fifth aspect, the present application provides a method for the biological production of bioethanol comprising the fermentative cultivation of a strain as provided in the first or fourth aspect above in a culture system comprising xylose.

Compared with wild Saccharomyces cerevisiae, the CE10 Saccharomyces cerevisiae strain provided in the first aspect of the application and the fermentation strain provided in the fourth aspect of the application can effectively utilize xylose for metabolism, so that the yield of bioethanol is improved; the CE10 saccharomyces cerevisiae strain and the fermentation strain provided in the fourth aspect of the application are adopted for fermentation culture in a culture system containing xylose, and can be used for large-scale production of ethanol.

In one possible embodiment, the concentration of xylose in the culture system is between 30 and 50g/L.

The concentration of xylose in a culture system is 30-50g/L, so that the strain provided by the application can be ensured to be well metabolized by utilizing xylose, and further more bioethanol can be produced. In one possible embodiment, the culture system further comprises glucose.

The culture system also contains glucose, so that the CE10 saccharomyces cerevisiae strain and the fermentation strain provided in the fourth aspect of the application can not only perform xylose metabolism, but also perform glucose metabolism, thereby being beneficial to producing more bioethanol.

In one possible embodiment, the concentration of glucose in the culture system is 70-100g/L.

The concentration of glucose in a culture system is 70-100g/L, so that the strain provided by the application can be well metabolized by glucose, and further more bioethanol can be produced.

In one possible embodiment, the temperature of the fermentation culture is 30-35 ℃, so that the CE10 saccharomyces cerevisiae strain and the fermentation strain provided in the fourth aspect of the application have good activity, and are beneficial to better metabolic production of bioethanol.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered limiting the scope, and that other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows a graph comparing xylose utilization after 48h fermentation for the strains provided in examples 1-5 of the present application.

FIG. 2 shows a graph comparing ethanol yields after 36h and 48h of fermentation, respectively, of the strains provided in examples 1-5 of the present application.

FIG. 3 shows a graph comparing acetic acid consumption rates after 48h fermentation of the strains provided in examples 1-5 of the present application.

FIG. 4 shows a graph comparing ethanol yields after 36h and 48h of fermentation respectively for the strains provided in example 2 and comparative examples 1-12 of the present application.

FIG. 5 shows a graph comparing ethanol yields after 36h and 48h of fermentation for the strains provided in examples 3-4 and comparative examples 13-17 of the present application, respectively.

FIG. 6 shows a graph comparing ethanol yields after 36h and 48h of fermentation for the strains provided in example 5 and comparative examples 18-23 of the present application, respectively.

Biological material preservation instructions:

biological material: CE10, class naming: saccharomyces cerevisiae Saccharomyces cerevisiae, deposited in China general microbiological culture Collection center (CGMCC) at a date of 2022, 05 and 09, has an address of: the collection number of the microbiological institute of China is CGMCC No.24860, and the national institute of sciences of China is No.1, no.3, north Chen West Lu, the Korean region of Beijing city.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The following provides a saccharomyces cerevisiae strain, a fermentation strain, a construction method and application thereof, and a method for producing bioethanol.

The application provides a Saccharomyces cerevisiae strain named CE10, and the preservation number of the Saccharomyces cerevisiae strain is CGMCC NO.24860.

Biological material: CE10, class naming: saccharomyces cerevisiae Saccharomyces cerevisiae, deposited in China general microbiological culture Collection center (CGMCC) at a date of 2022, 05 and 09, has an address of: the collection number of the microbiological institute of China is CGMCC No.24860, and the national institute of sciences of China is No.1, no.3, north Chen West Lu, the Korean region of Beijing city.

The CE10 saccharomyces cerevisiae strain is obtained through the adaptive evolution of the wild saccharomyces cerevisiae strain for a plurality of years, and compared with the wild saccharomyces cerevisiae, the CE10 saccharomyces cerevisiae strain provided by the application can effectively utilize xylose for metabolism, so that the yield of bioethanol is improved; the tolerance of the strain to acid inhibitors such as acetic acid and the like can be improved, and the yield of bioethanol can be further improved.

The culture conditions of the CE10 Saccharomyces cerevisiae strain were: the single colony of CE10 Saccharomyces cerevisiae strain was picked up and cultured in YPD medium at 30℃under shaking at 200 rpm.

The preservation conditions of the CE10 Saccharomyces cerevisiae strain were: inoculating CE10 Saccharomyces cerevisiae into YPD solid plate culture medium, culturing at 30deg.C for 48-72 hr, and preserving at 4deg.C for a short period. Or inoculating the cultured CE10 Saccharomyces cerevisiae strain solution into 80% (v/v) sterile glycerol cryopreservation tube, and freezing at-80deg.C for long-term storage.

The application also provides a construction method of the fermentation strain, which comprises the step of integrating a target gene into the genome of the CE10 saccharomyces cerevisiae strain. The target gene is selected from MSN2, TIPI, YRB1 or SPII. Wherein, the MSN2 has a sequence shown as SEQ ID NO.1, the TIPI has a sequence shown as SEQ ID NO.2, the YRB1 has a sequence shown as SEQ ID NO.3, and the SPII has a sequence shown as SEQ ID NO. 4.

The target genes MSN2 (transcription factor), TIP1 (model gene), YRB1 (model gene) or SPI1 (cell wall gene) are respectively integrated into the genome of the CE10 saccharomyces cerevisiae strain to construct a fermentation strain, compared with wild saccharomyces cerevisiae, xylose can be effectively utilized for metabolism, the yield of bioethanol is further improved, and the tolerance of the strain to acid inhibitors such as acetic acid can be remarkably improved; compared with the CE10 saccharomyces cerevisiae strain, the fermentation strain constructed by the method can further improve the utilization efficiency of the strain on xylose and the yield of bioethanol.

The application also provides a construction method of the fermentation strain, which comprises the steps of firstly deleting the CAT8 gene in the genome of the CE10 saccharomyces cerevisiae strain, and integrating the target gene into the genome of the CE10 saccharomyces cerevisiae strain with the CAT8 gene in the genome of the CE10 saccharomyces cerevisiae strain as an integration site. The target gene is selected from MSN2, TIPI, YRB1 or SPII. Wherein, the MSN2 has a sequence shown as SEQ ID NO.1, the TIPI has a sequence shown as SEQ ID NO.2, the YRB1 has a sequence shown as SEQ ID NO.3, and the SPII has a sequence shown as SEQ ID NO. 4.

Firstly, CAT8 genes in the genome of the saccharomyces cerevisiae strain CE10 are deleted, so that metabolic pathways of the subsequently prepared fermentation strain during fermentation flow to the ethanol fermentation direction more, and the yield of bioethanol is improved. And then taking CAT8 as an integration site, integrating MSN2, TIP1, YRB1 or SPI1 target genes into the genome of the Saccharomyces cerevisiae strain with the CAT8 genes deleted to construct a fermentation strain, wherein the constructed fermentation strain can remarkably improve the utilization rate of the strain to xylose and the tolerance of the strain to acid inhibitors such as acetic acid, and further improve the yield of bioethanol.

Further, the step of integrating the gene of interest into the genome of the Saccharomyces cerevisiae strain deleted for CAT8 gene comprises: the linear fragment containing the gene of interest is integrated into the genome of the Saccharomyces cerevisiae strain deleted for CAT8 gene and the gene of interest is overexpressed.

CAT8 is used as an integration site and the target gene is over-expressed, so that stable fermentation strains can be constructed.

In an embodiment of the present application, a method for preparing a linear fragment containing a gene of interest includes: the vector fragment and the target gene fragment were first digested and ligated by Golden Gate Assembly (GGA) to construct a plasmid containing the target gene. The plasmid containing the target gene was electrotransferred into E.coli competence, and positive clone colonies were selected in solid LB medium with kanamycin resistance. A certain number of positive clone colonies are selected, whether correct bands are amplified or not is confirmed through colony PCR, and if the sizes of the bands and the sequencing results are correct, corresponding plasmids can be extracted. And amplifying the linear fragment containing the target gene by using the extracted plasmid as a template.

In the examples herein, the gene of interest was integrated into the genome of the CE10 s.cerevisiae strain using the principle of homologous recombination, such that the gene of interest was overexpressed. The linear fragment containing the target gene and the other two linear fragments having homologous fragments between the linear fragments containing the target gene are integrated together into the CAT8 site of the CE10 s.cerevisiae strain by a lithium acetate transformation method.

The application also provides a fermentation strain, which is prepared by adopting the construction method of the fermentation strain.

Compared with wild Saccharomyces cerevisiae, the fermentation strain provided by the application can effectively utilize xylose for metabolism; compared with the CE10 saccharomyces cerevisiae strain, the utilization efficiency of the strain to xylose and the tolerance of the strain to acid inhibitors such as acetic acid can be further effectively improved, and the yield of bioethanol is further improved.

In the application, when a method of deleting CAT8 gene in the genome of the CE10 saccharomyces cerevisiae strain firstly, then integrating a target gene fragment into the genome of the CE10 saccharomyces cerevisiae strain deleted with CAT8 gene by using a homologous recombination principle and over-expressing the target gene by taking CAT8 in the genome of the CE10 saccharomyces cerevisiae strain as an integration site is adopted to construct a fermentation strain, when the target gene is MSN2, the fermentation strain is named as WXY246; when the target gene is TIP1, the fermentation strain is named WXY247; when the target gene is YRB1, the fermentation strain is named WXY249; when the gene of interest was SPI1, the fermenting strain was designated WXY256.

Compared with wild Saccharomyces cerevisiae, the WXY246 fermentation strain, the WXY247 fermentation strain, the WXY249 fermentation strain and the WXY256 fermentation strain provided by the application can effectively utilize xylose for metabolism, so that the yield of bioethanol is improved, and the tolerance of the strain to acid inhibitors such as acetic acid can be obviously improved; compared with the CE10 saccharomyces cerevisiae strain, the utilization efficiency of the strain to xylose, the tolerance to acid inhibitors such as acetic acid and the like and the yield of bioethanol can be further improved.

Wherein, the culture conditions of the WXY246 fermentation strain are as follows: single colony bacteria of the WXY246 fermentation strain were picked up in YPD medium and cultured at 30℃under shaking at 200 rpm.

The storage conditions of the WXY246 fermentation strain were: WXY246 fermentation bacteria were inoculated into YPD solid plate medium, cultured at 30℃for 48-72 hours, and then preserved at 4℃for a short period of time. Or inoculating the cultured WXY246 zymocyte liquid into 80% (v/v) sterile glycerol freezing tube, and freezing at-80deg.C for long-term storage.

Wherein, the culture conditions of the WXY247 fermentation strain are as follows: single colony bacteria of the WXY247 fermentation strain are selected and cultured in YPD medium at 30 ℃ under shaking at 200 rpm.

The storage conditions of the WXY247 fermentation strain are as follows: WXY247 fermentation bacteria are inoculated to YPD solid plate culture medium, cultured at 30 ℃ for 48-72 hours and preserved at 4 ℃ for a short period. Or inoculating the cultured WXY247 zymocyte liquid into 80% (v/v) sterile glycerol freezing tube, and freezing at-80deg.C for long-term storage.

Wherein, the culture conditions of the WXY249 fermentation strain are as follows: single colony bacteria of the WXY249 fermentation strain were picked up in YPD medium and cultured at 30℃under shaking at 200 rpm.

The storage conditions of the WXY249 fermentation strain were: WXY249 fermentation broth was inoculated into YPD solid plate medium, cultured at 30℃for 48-72 hours, and then stored at 4℃for a short period of time. Or inoculating the cultured WXY249 zymocyte liquid into 80% (v/v) sterile glycerol freezing tube, and freezing at-80deg.C for long-term storage.

Wherein, the culture conditions of the WXY256 fermentation strain are as follows: single colony bacteria of the WXY256 fermentation strain are picked up and cultured in YPD medium at 30 ℃ under shaking at 200 rpm.

The storage conditions of the WXY256 fermentation strain were: WXY256 fermentation broth was inoculated into YPD solid plate medium, cultured at 30℃for 48-72 hours, and then preserved at 4℃for a short period of time. Or inoculating the cultured WXY256 zymocyte liquid into 80% (v/v) sterile glycerol freezing tube, and freezing at-80deg.C for long-term storage.

The application also provides a method for biologically producing bioethanol, which comprises the steps of fermenting and culturing the CE10 saccharomyces cerevisiae strain and the fermentation strain provided by the application in a culture system containing xylose.

Compared with wild Saccharomyces cerevisiae, the CE10 Saccharomyces cerevisiae strain and the constructed fermentation strain provided by the application can effectively utilize xylose for metabolism, so that the yield of bioethanol is improved, and the CE10 Saccharomyces cerevisiae strain and the constructed fermentation strain are adopted for fermentation culture in a culture system containing xylose, so that the CE10 Saccharomyces cerevisiae strain and the constructed fermentation strain can be used for mass production of ethanol.

Furthermore, the concentration of xylose in a culture system is 30-50g/L, so that the strain provided by the application can be well metabolized by utilizing xylose, and further more bioethanol can be produced. As an example, the concentration of xylose in the culture system may be 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, or the like.

In the embodiment of the application, the culture system also contains glucose, so that the CE10 saccharomyces cerevisiae strain and the fermentation strain constructed by the application can not only perform xylose metabolism, but also perform glucose metabolism, and further the production of more bioethanol is facilitated.

Further, the concentration of glucose in a culture system is 70-100g/L, so that the strain provided by the application can be ensured to be well metabolized by glucose, and further more bioethanol can be produced. As an example, the concentration of glucose in the culture system may be 70g/L, 75g/L, 80g/L, 90g/L, 100g/L, or the like.

Still further, the temperature of fermentation culture is 30-35 ℃, so that the CE10 saccharomyces cerevisiae strain and the fermentation strain constructed by the method have good activity, and the bioethanol can be produced better by metabolism. As an example, the temperature of the fermentation culture may be 30 ℃, 32 ℃, 35 ℃, or the like.

Example 1

The embodiment provides a CE10 Saccharomyces cerevisiae strain (preservation number is CGMCC NO. 24860).

Example 2

The present example provides a WXY246 fermenting strain, constructed by the following steps:

(1) Amplifying the linear fragment containing the gene of interest.

T1-0 with the sequence shown as SEQ ID NO.5 is used as a template, T1-0-gga-F with the sequence shown as SEQ ID NO.6 and T1-0-gga-R with the sequence shown as SEQ ID NO.7 are respectively used as upstream and downstream primers, and a linear T1-0 carrier fragment is amplified by Polymerase Chain Reaction (PCR). The MSN2 target gene fragment with the sequence shown as SEQ ID NO.1 is amplified by PCR by taking the rough extracted wild saccharomyces cerevisiae genome as a template and taking MSN2-gga-F with the sequence shown as SEQ ID NO.8 and MSN2-gga-R with the sequence shown as SEQ ID NO.9 as upstream and downstream primers respectively.

Determining whether the band size of the amplified MSN2 target gene fragment is correct or not through agarose gel electrophoresis, and comparing the base sequences of the amplified MSN2 target gene fragment through sequencing. If the band size and the gene sequence are correct, the plasmid T1-0-MSN2 is constructed by carrying out enzyme digestion and connection on the obtained T1-0 carrier fragment and the MSN2 target gene fragment by a GGA method. Wherein, in the enzyme digestion and ligation process, the endonuclease used is BsaI-HFv 2, and the Ligase used is T4 DNA Ligase.

Plasmid T1-0-MSN2 was electrotransferred into E.coli competence and positive clone colonies were selected with solid LB medium with kanamycin resistance. A certain number of positive clone colonies are selected, whether correct bands are amplified or not is confirmed through colony PCR, and if the sizes of the bands and the sequencing results are correct, it is determined that the T1-0-MSN2 plasmid can be extracted. The extracted T1-0-MSN2 plasmid is used as a template, L1-F with a sequence shown as SEQ ID NO.10 and L2-R with a sequence shown as SEQ ID NO.11 are respectively used as an upstream primer and a downstream primer, and the linear fragment of L1-pPGK1-MSN2-tPGI1-L2 is amplified. Wherein the step of electrotransferring the plasmid T1-0-MSN2 to E.coli competence comprises: the electric rotating cup is cleaned by 75 percent alcohol, and is reversely buckled on dust-free paper for airing, and the electric rotating cup is inserted on ice paved with a plastic film for precooling for 20 minutes. The electroreceptive cells were removed from the freezer at-80℃and were inserted onto ice for thawing. 2.5 mu L of the connection product T1-0-MSN2 is sucked and added into 50 mu L of electric transfer escherichia coli competence to be mixed uniformly, and the mixed sample is added into the aperture of an electric transfer cup and is inserted into an electric transfer instrument to be subjected to pulse electric shock. 200. Mu.L of antibiotic-free LB was added to the shocked sample, and the mixture was blown and mixed well, and transferred to a 1.5mL centrifuge tube. Culturing the electric-transformed bacterial liquid in a shaking table at 37 ℃ and 200rpm for 1h, centrifuging at 12000rpm for 2min, collecting bacterial cells, uniformly mixing 50 mu L of liquid, coating the liquid on a corresponding resistance plate, and culturing in an inverted manner in a culture box at 37 ℃ for 16h.

(2) CAT8UP-G418-L1 and L2-CAT8DOWN, in which homologous fragments exist to linear fragments containing the target gene, were amplified.

The linear fragment CAT8UP-G418-L1 is amplified by taking T2-CAT8UP-G418-L1 with a sequence shown as SEQ ID NO.12 as a template, taking CAT8UP-F with a sequence shown as SEQ ID NO.13 and L1-R with a sequence shown as SEQ ID NO.14 as upstream and downstream primers respectively.

The linear fragment L2-CAT8DOWN is amplified by using T3-L2-CAT8DOWN with the sequence shown as SEQ ID NO.15 as a template, and L2-F with the sequence shown as SEQ ID NO.16 and CAT8DOWN-R with the sequence shown as SEQ ID NO.17 as upstream and downstream primers respectively.

(3) A linear fragment containing the gene of interest, CAT8UP-G418-L1 and L2-CAT8DOWN were homologously recombined into the genome of the CE10 s.cerevisiae strain.

The CAT8 gene of the genome of the CE10 Saccharomyces cerevisiae strain is deleted, CAT8 is taken as an integration site, and linear fragments L1-pPGK1-MSN2-tPGI1-L2, CAT8UP-G418-L1 and L2-CAT8DOWN containing the target gene are transformed into the CE10 Saccharomyces cerevisiae strain by a Saccharomyces cerevisiae lithium acetate transformation method so as to enable the MSN2 gene to be over-expressed. The method for converting the saccharomyces cerevisiae lithium acetate comprises the following steps of: 20mL of YPD liquid medium and single colony of CE10 s.cerevisiae strain were added to the flask, and cultured in a shaker at 30℃and 200rpm for 12 hours to obtain a bacterial liquid. Adding bacterial liquid into 20mLYPD liquid medium, and OD ₆₀₀ Adjusting to 0.2, culturing in shaking table at 30deg.C and 200rpm for 4 hr to obtain OD of the cultured bacterial liquid ₆₀₀ 0.7. 1mLOD was added to a 1.5mL centrifuge tube ₆₀₀ The bacterial liquid is 0.7, the bacterial liquid is centrifuged for 2min at 3000g at 25 ℃, and the supernatant is discarded. 1mLddH is sucked up ₂ O water washing bacteria liquid, 3000g centrifuging for 2min, and discarding supernatant. Re-pipetting 1ml ddH ₂ O water washing bacteria liquid, 3000g centrifuging for 2min, and discarding supernatant. The washed cells were resuspended in 100. Mu.L of 0.1MLiAc solution, cultured in an incubator at 30℃for 10min, centrifuged at 3000g for 2min, and the supernatant was discarded. Simultaneously, the ssDNA solution is placed on a heating block at 100 ℃ and heated for 10min, and immediately inserted into ice after the heating is finished. The cells were resuspended with 20. Mu.L of ddH2O, then 360. Mu.L of 50% PEG solution, 55. Mu.L of 1MLiAc solution, 75. Mu.L of treated ssDNA solution and a suspension containing 1500ng of L1-pPGK1-MSN2-tPGI1-L2, 1500ng of CAT8UP-G418-L1 and 1500ng of L2-CAT8DOWNddH were sequentially added ₂ And 40. Mu.L of the mixed solution of O. Shaking the mixture at 1400rpm with vigorous vortex until completely mixed, culturing in 30 deg.C incubator for 30min, and adding water in 42 deg.C water bathBath for 30min. After completion of the water bath, 7500g of the cells were centrifuged for 2 minutes, and the centrifuged cells were resuspended in 1mLYPD liquid medium and cultured in a shaker at 30℃and 200rpm for 3 hours. Centrifuge at 3000g for 2min, discard supernatant. 1mL of ddH was added ₂ Washing the thalli with O water, centrifuging for 2min at 3000g, and discarding the supernatant. 1mL of ddH was added ₂ Washing the thalli with O water, centrifuging for 2min at 3000g, and discarding the supernatant. The cells were resuspended in 50. Mu.L of the solution and plated on a resistant solid medium and cultured upside down in a 30℃incubator for 4 days.

Example 3

This example provides a WXY247 strain, which is constructed in a manner substantially similar to that of example 2, except that: the target gene in this example is TIP1, and the amplification of the target gene fragment of TIP1 is carried out by the following steps: the wild saccharomyces cerevisiae genome which is subjected to rough extraction is used as a template, TIP1-gga-F with a sequence shown as SEQ ID NO.18 and TIP1-gga-R with a sequence shown as SEQ ID NO.19 are respectively used as upstream and downstream primers, and a TIP1 target gene fragment with a sequence shown as SEQ ID NO.2 is amplified by PCR.

Example 4

This example provides a WXY 249-fermenting strain, and the construction method of the WXY 249-fermenting strain is basically similar to that of example 2, except that: the target gene in this example is YRB1, and the amplification of the YRB1 target gene fragment is performed as follows: the gene fragment of YRB1 with the sequence shown in SEQ ID NO.3 is amplified by PCR by taking the rough extracted wild saccharomyces cerevisiae genome as a template and taking YRB1-gga-F with the sequence shown in SEQ ID NO.20 and YRB1-gga-R with the sequence shown in SEQ ID NO.21 as upstream and downstream primers respectively.

Example 5

The present example provides a WXY256 fermentation strain (the construction method of the WXY256 fermentation strain is basically similar to that of example 2, except that the target gene in this example is SPI1, and the SPI1 target gene fragment is amplified by PCR using a crude wild-type Saccharomyces cerevisiae genome as a template, SPI1-gga-F as shown in SEQ ID NO.22 and SPI1-gga-R as shown in SEQ ID NO.23 as upstream and downstream primers, respectively.

Comparative examples 1 to 23

Comparative examples 1 to 23 each provide a fermentation strain, which was constructed in a manner substantially similar to that of example 2, except that: the target genes are different, and the upstream and downstream primers for amplifying the target gene fragments are shown in Table 1.

TABLE 1

Experimental example 1

The strain provided in examples 1-5 was subjected to a mixed sugar fermentation experiment, the xylose utilization results after 48h of strain fermentation are shown in FIG. 1, the ethanol yield results after 36h and 48h of strain fermentation are shown in FIG. 2, and the acetic acid consumption results after 48h of strain fermentation are shown in FIG. 3. Wherein, the steps of the mixed sugar fermentation experiment are as follows: the cultured bacterial liquid is added into a fermentation culture medium, OD600 is regulated to 1, and the bacterial liquid is cultured in a shaking table at 30 ℃ and 200rpm for 48 hours in dark. The preparation steps of the fermentation medium are as follows: 80g of peptone, 10g of yeast extract, 80g of glucose, 40g of xylose and 3g of acetic acid were added to 1L of ddH ₂ O, a fermentation medium is obtained.

As can be seen from FIG. 1, the strains of examples 1 to 5 each efficiently utilized xylose for metabolism; as the wild type saccharomyces cerevisiae cannot metabolize by xylose, the strains provided in examples 1-5 can significantly improve the utilization efficiency of xylose relative to the wild type saccharomyces cerevisiae. And the xylose utilization efficiency of the strain provided in examples 2-4 after 48h fermentation is higher than that of the strain provided in example 1 after 48h fermentation, which indicates that the utilization efficiency of the strain on xylose can be further improved by integrating MSN2, TIP1, YRB1 or SPI1 into the genome of the CE10 Saccharomyces cerevisiae strain.

As can be seen from FIG. 2, the ethanol yields of the strains provided in example 1 after 36h and 48h of fermentation were 0.412g/g total sugar and 0.439g/g total sugar, respectively, reaching 80.8% and 86.1% of the theoretical maximum, respectively, indicating that the strains provided in example 1 have good bioethanol production capacity. Further, the ethanol yields of the strains of examples 2-5 after 36h and 48h fermentation were higher than those of the strain of example 1 after 36h and 48h fermentation, indicating that integration of MSN2, TIP1, YRB1 or SPI1 into the genome of the CE10 Saccharomyces cerevisiae strain could further increase bioethanol yield.

As can be seen from FIG. 3, the strains of examples 1 to 5 each consume acetic acid efficiently, indicating that the strains provided in examples 1 to 5 have a certain tolerance to acetic acid. And the acetic acid consumption rate of the strain provided in examples 2-5 after 48h fermentation is higher than that of the strain provided in example 1 after 48h fermentation, which indicates that the WXY246 fermentation strain, the WXY247 fermentation strain, the WXY249 fermentation strain or the WXY256 fermentation strain provided in the application can further improve the tolerance of the strain to acetic acid compared with the CE10 saccharomyces cerevisiae strain, and shows that the integration of MSN2, TIP1, YRB1 or SPI1 into the genome of the CE10 saccharomyces cerevisiae strain can further improve the tolerance of the strain to acetic acid.

Experimental example 2

The results of ethanol production after 36h and 48h of fermentation of the strain provided in example 2 and comparative examples 1 to 12 are shown in FIG. 4. Among them, the mixed sugar fermentation experiment was the same as in experimental example 1.

As can be seen from FIG. 4, the ethanol yields of the strains of example 2 after 36h and 48h fermentation are significantly higher than those of the strains of comparative examples 1-12 after 36h and 48h fermentation, indicating that the MSN2 transcription factor integration of example 2 into the genome of the CE10 Saccharomyces cerevisiae strain is effective in improving bioethanol yield as compared to the transcription factors employed in comparative examples 1-12.

Experimental example 3

The results of the ethanol production after 36h and 48h fermentation of the strains provided in examples 3 to 4 and comparative examples 13 to 17 are shown in FIG. 5. Among them, the mixed sugar fermentation experiment was the same as in experimental example 1.

The genes of interest in examples 3-4 and comparative examples 13-17 are all the same expression pattern genes, and as can be seen from FIG. 5, the ethanol yields of the strains of examples 3-4 after 36h and 48h fermentation are significantly higher than those of the strains of comparative examples 13-17 after 36h and 48h fermentation, indicating that the integration of the same expression pattern genes TIP1 and YRB1 respectively selected in examples 3 and 4 into the genome of the CE10 Saccharomyces cerevisiae strain is effective in improving bioethanol yield as compared to the same expression pattern genes employed in comparative examples 13-17.

Experimental example 4

The results of ethanol production after 36h and 48h of fermentation of the strain provided in example 5 and comparative examples 18 to 23 are shown in FIG. 6. Among them, the mixed sugar fermentation experiment was the same as in experimental example 1.

As can be seen from FIG. 6, the ethanol yields of the strains of example 5 after 36h and 48h are significantly higher than those of the strains of comparative examples 18-23 after 36h and 48h, indicating that the integration of the cell wall gene SPI1 selected in example 5 into the genome of the CE10 Saccharomyces cerevisiae strain is effective in improving bioethanol yield as compared to the cell wall gene employed in comparative examples 18-23.

In conclusion, compared with wild Saccharomyces cerevisiae, the CE10 Saccharomyces cerevisiae strain, the WXY246 fermentation strain, the WXY247 fermentation strain, the WXY249 fermentation strain or the WXY256 fermentation strain provided by the application can remarkably improve the xylose utilization efficiency and the bioethanol yield, so that the CE10 Saccharomyces cerevisiae strain, the WXY246 fermentation strain, the WXY247 fermentation strain or the WXY256 fermentation strain has a good application prospect in the bioethanol production field.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

SEQUENCE LISTING

<110> university of capital and education

<120> Saccharomyces cerevisiae strain, fermentation strain, construction method thereof and bioethanol production method

<130> 2022.06.21

<160> 69

<170> PatentIn version 3.5

<210> 1

<211> 2115

<212> DNA

<213> Synthesis

<400> 1

atgacggtcg accatgattt caatagcgaa gatattttat tccccataga aagcatgagt 60

agtatacaat acgtggagaa taataaccca aataatatta acaacgatgt tatcccgtat 120

tctctagata tcaaaaacac tgtcttagat agtgcggatc tcaatgacat tcaaaatcaa 180

gaaacttcac tgaatttggg gcttcctcca ctatctttcg actctccact gcccgtaacg 240

gaaacgatac catccactac cgataacagc ttgcatttga aagctgatag caacaaaaat 300

cgcgatgcaa gaactattga aaatgatagt gaaattaaga gtactaataa tgctagtggc 360

tctggggcaa atcaatacac aactcttact tcaccttatc ctatgaacga cattttgtac 420

aacatgaaca atccgttaca atcaccgtca ccttcatcgg tacctcaaaa tccgactata 480

aatcctccca taaatacagc aagtaacgaa actaatttat cgcctcaaac ttcaaatggt 540

aatgaaactc ttatatctcc tcgagcccaa caacatacgt ccattaaaga taatcgtctg 600

tccttaccta atggtgctaa ttcgaatctt ttcattgaca ctaacccaaa caatttgaac 660

gaaaaactaa gaaatcaatt gaactcagat acaaattcat attctaactc catttctaat 720

tcaaactcca attctacggg taatttaaat tccagttatt ttaattcact gaacatagac 780

tccatgctag atgattacgt ttctagtgat ctcttattga atgatgatga tgatgacact 840

aatttatcac gccgaagatt tagcgacgtt ataacaaacc aatttccgtc aatgacaaat 900

tcgaggaatt ctatttctca ctctttggac ctttggaacc atccgaaaat taatccaagc 960

aatagaaata caaatctcaa tatcactact aattctacct caagttccaa tgcaagtccg 1020

aataccacta ctatgaacgc aaatgcagac tcaaatattg ctggcaaccc gaaaaacaat 1080

gacgctacca tagacaatga gttgacacag attcttaacg aatataatat gaacttcaac 1140

gataatttgg gcacatccac ttctggcaag aacaaatctg cttgcccaag ttcttttgat 1200

gccaatgcta tgacaaagat aaatccaagt cagcaattac agcaacagct aaaccgagtt 1260

caacacaagc agctcacctc gtcacataat aacagtagca ctaacatgaa atccttcaac 1320

agcgatcttt attcaagaag gcaaagagct tctttaccca taatcgatga ttcactaagc 1380

tacgacctgg ttaataagca ggatgaagac cccaagaacg atatgctgcc gaattcaaat 1440

ttgagttcat ctcaacaatt tatcaaaccg tctatgattc tttcagacaa tgcgtccgtt 1500

attgcgaaag tggcgactac aggcttgagt aatgatatgc catttttgac agaggaaggt 1560

gaacaaaatg ctaattctac tccaaatttc gatctttcca tcactcaaat gaatatggct 1620

ccattatcgc ctgcatcatc atcctccacg tctcttgcaa caaatcattt ctatcaccat 1680

ttcccacagc agggtcacca taccatgaac tctaaaatcg gttcttccct tcggaggcgg 1740

aagtctgctg tgcctttgat gggtacggtg ccgcttacaa atcaacaaaa taatataagc 1800

agtagtagtg tcaactcaac tggcaatggt gctggggtta cgaaggaaag aaggccaagt 1860

tacaggagaa aatcaatgac accgtccaga agatcaagtg tcgtaataga atcaacaaag 1920

gaactcgagg agaaaccgtt ccactgtcac atttgtccca agagctttaa gcgcagcgaa 1980

catttgaaaa ggcatgtgag atctgttcac tctaacgaac gaccatttgc ttgtcacata 2040

tgcgataaga aatttagtag aagcgataat ttgtcgcaac acatcaagac tcataaaaaa 2100

catggagaca tttaa 2115

<210> 2

<211> 633

<212> DNA

<213> Synthesis

<400> 2

atgtccgttt ccaagattgc tttcgtttta agtgccattg cctctttggc cgtcgctgac 60

accagcgccg ccgaaactgc tgaattgcaa gctattatcg gtgacatcaa ctctcatctt 120

tctgactact tgggtctaga aactggcaac agtggattcc aaattccatc tgatgtcttg 180

agtgtgtatc aacaagtcat gacttacacc gatgacgctt acactacctt gtttagtgaa 240

ttggactttg atgctatcac taagacaatt gttaaattgc catggtacac cacaagattg 300

agttctgaaa tcgctgctgc tcttgcctcc gtttccccag cttcttccga ggctgcatct 360

tcttccgagg ctgcatcttc ttccaaggct gcatcttctt ccgaagctac atcctctgcc 420

gctccatcct cttctgctgc cccatcttct tctgctgccc catcatcatc tgccgaatca 480

tcttctaagg ccgtttcttc ttctgtcgct ccaactacct cttctgtcag cacttctaca 540

gtcgaaactg cttccaatgc cggtcaaaga gtcaatgcag gcgctgcctc tttcggtgct 600

gttgttgcag gtgcagctgc tttattgtta taa 633

<210> 3

<211> 606

<212> DNA

<213> Synthesis

<400> 3

atgtctagcg aagataagaa acctgtcgtc gacaagaagg aagaggctgc tccaaagcca 60

ccatcctctg ctgtcttctc catgtttggt ggtaagaagg ccgaaaagcc agaaaccaag 120

aaagacgaag aagataccaa ggaggaaacc aagaaggaag gtgatgatgc tccagaatca 180

ccagatatcc attttgaacc agtggttcac ctggaaaagg tagatgttaa gacaatggaa 240

gaagacgaag aagttcttta caaggtcaga gccaagcttt tcagattcga tgccgatgcc 300

aaggaatgga aagaaagagg tactggtgac tgtaagttct tgaagaacaa aaagactaac 360

aaggttagaa tattgatgag aagagacaag accttaaaga tttgtgctaa ccacatcatt 420

gctccagaat acactttgaa gcctaacgtt ggttctgata gatcttgggt gtatgcttgt 480

acagcagata ttgcagaagg tgaagcagaa gccttcactt ttgctatcag atttggcagt 540

aaggaaaatg ctgataaatt taaagaagaa tttgaaaaag ctcaagaaat caacaaaaag 600

gcttag 606

<210> 4

<211> 447

<212> DNA

<213> Synthesis

<400> 4

atgttgtcta acgctaagct ccttctatca ttggccatgg cctctacggc tctcggattg 60

gtatctaatt ctagttcctc tgtaatcgtg gtaccatcaa gcgatgctac tattgccggt 120

aacgatacag ccacgccagc accagagcca tcatccgccg ctccaatatt ctacaactcg 180

actgctactg caacacagta cgaagttgtc agtgaattca ctacttactg cccagaacca 240

acgactttcg taacgaatgg cgctacattc actgttactg ccccaactac gttaacaatt 300

accaactgtc cttgcactat cgagaagcct acttcagaaa catcggtttc ttctacacat 360

gatgtggaga caaattctaa tgctgctaac gcaagagcaa tcccaggagc cctaggtttg 420

gctggtgcag ttatgatgct tttatga 447

<210> 5

<211> 4123

<212> DNA

<213> Synthesis

<400> 5

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatggaac 420

ggatcccgtc tcccccggtc cgtttgttct atacttctct ctgctatacc tacaagcaag 480

gtaatcggaa gtagtattac gcaggaatat cccgcgcgaa gctacaattt ttggactcca 540

acgtcaaagc aggggagtca gaagtcccct ctaaaattgc ctaggtacgc acagatatta 600

taacatctgc acaataggca tttgcaagaa ttactcgtga gtaaggaaag agtgaggaac 660

tatcgcatac ctgcatttaa agatgccgat ttgggcgcga atcctttatt ttggcttcac 720

cctcatacta ttatcagggc cagaaaaagg aagtgtttcc ctccttcttg aattgatgtt 780

accctcataa agcacgtggc ctcttatcga gaaagaaatt accgtcgctc gtgatttgtt 840

tgcaaaaaga acaaaactga aaaaacccag acacgctcga cttcctgtct tcctattgat 900

tgcagcttcc aatttcgtca cacaacaagg tcctagcgac ggctcacagg ttttgtaaca 960

agcaatcgaa ggttctggaa tggcgggaaa gggtttagta ccacatgcta tgatgcccac 1020

tgtgatctcc agagcaaagt tcgttcgatc gtactgttac tctctctctt tcaaacagaa 1080

ttgtccgaat cgtgtgacaa caacagcctg ttctcacaca ctcttttctt ctaaccaagg 1140

gggtggttta gtttagtaga acctcgtgaa acttacattt acatatatat aaacttgcat 1200

aaattggtca atgcaagaaa tacatatttg gtcttttcta attcgtagtt tttcaagttc 1260

ttagatgctt tctttttctc ttttttacag atcatcaagg aagtaattat ctacttttta 1320

caacaaatat aaaacaaggt atcgaacaaa tcgctcttaa atatatacct aaagaacatt 1380

aaagctatat tataagcaaa gatacgtaaa ttttgcttat attattatac acatatcata 1440

tttctatatt tttaagattt ggttatataa tgtacgtaat gcaaaggaaa taaattttat 1500

acattattga acagcgtcca agtaactaca ttatgtgcac taatagttta gcgtcgtgaa 1560

gactttattg tgtcgcgaaa agtaaaaatt ttaaaaatta gagcaccttg aacttgcgaa 1620

aaaggttctc atcaactgtt taaaaggagg atatcaggtc ctatttctga caaacaatat 1680

acaaatttag tttcaaagat gaatcagtgc gcgaaggaca taactcatga agcctccagt 1740

ataccgacaa agcgccaagg aactgtaata tatagctacg ccctatctgg acgattgggc 1800

gacttttacg tacggttgct caattcctac gcaacttaat atattttgca acggttaaat 1860

cggcttgaag ctcgggctat ccaactcgcg gactagagac gtctagagtt ccatcccaat 1920

ggcgcgccga gcttggctcg agcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 1980

ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 2040

tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 2100

ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 2160

gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 2220

gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 2280

ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 2340

ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 2400

cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 2460

ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 2520

tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 2580

gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 2640

tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 2700

gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 2760

tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 2820

ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 2880

agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 2940

gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 3000

attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 3060

agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta gaaaaactca 3120

tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga 3180

aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga 3240

tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 3300

tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 3360

aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg 3420

tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga 3480

cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc 3540

aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc 3600

tggaatgctg ttttcccagg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg 3660

ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc 3720

tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca 3780

tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc 3840

catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac 3900

gtttcccgtt gaatatggct catactcttc ctttttcaat attattgaag catttatcag 3960

ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 4020

gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg 4080

acattaacct ataaaaatag gcgtatcacg aggccctttc gtc 4123

<210> 6

<211> 45

<212> DNA

<213> Synthesis

<400> 6

accaggtctc aatcgaacaa atcgctctta aatatatacc taaag 45

<210> 7

<211> 53

<212> DNA

<213> Synthesis

<400> 7

accaggtctc aaccttgttt tatatttgtt gtaaaaagta gataattact tcc 53

<210> 8

<211> 36

<212> DNA

<213> Synthesis

<400> 8

accaggtctc aaggtatgac ggtcgaccat gatttc 36

<210> 9

<211> 46

<212> DNA

<213> Synthesis

<400> 9

accaggtctc acgatttaaa tgtctccatg ttttttatga gtcttg 46

<210> 10

<211> 28

<212> DNA

<213> Synthesis

<400> 10

cgtctccccc ggtccgtttg ttctatac 28

<210> 11

<211> 27

<212> DNA

<213> Synthesis

<400> 11

gcggacttag tccgtttctc ggctatc 27

<210> 12

<211> 5656

<212> DNA

<213> Synthesis

<400> 12

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatggaac 420

ggatcccgtc tcgacaaagc gccaaggaac tgtaatatat agctacgccc tatctggacg 480

attgggcgac ttttacgtac ggttgctcaa ttcctacgca acttaatata ttttgcaacg 540

gttaaatcgg cttgaagctc gggctatcca actcgcggac tatcggcatg ccggtagagg 600

tgtggtcaat aagagcgacc tcatgctata cctgagaaag caacctgacc tacaggaaag 660

agttactcaa gaataagaat tttcgtttta aaacctaaga gtcactttaa aatttgtata 720

cacttatttt ttttataact aggtggatga tgaactactt tccctcactg aaattaaaga 780

gcttttacac ctttttttca aattttggtc taatcaggta ccaattctaa acaatgacca 840

cttcttaata tatttcaaca attttgttga agtcgtgaag catttatcta cagaaaattt 900

ggagacgaat aacactacta aaagtacagt caccactaat catgaaatat ttgccctaaa 960

gcttctgatg atgttacaaa tggggttact cgttaagatt aaaatggaaa aaataaaata 1020

cactgtaccg aagaatccga aggccaaata tgctagattg atggcatatt atcatcagct 1080

ttccctaata attcctaaaa atccttattt tttaaatatg tccacaactt cattaccatc 1140

cttacaattg ttatctttgg catcctttta ttatttaaac gtgggtgata tttcggcaat 1200

ctatggggta agaggccgta ttgtttctat ggcacaacaa ttgagacttc acagatgtcc 1260

tagtgccgta ttaagtgtac actctaaccc agttctacaa aaatttgagc aaagcgagag 1320

gagattactg ttttgggcaa tttattacgt tgacgttttt gcctcattgc aacttggtgt 1380

tcctcgttta cttaaggatt ttgatattga atgtgcatta ccaatttctg atgttgagta 1440

taagcatcga tctgtttagc ttgcctcgtc cccgccgggt cacccggcca gcgacatgga 1500

ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg 1560

tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca tacattttga 1620

tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg agcagggaaa 1680

cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa 1740

aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg 1800

atacagttct cacatcacat ccgaacataa acaaccatgg gtaaggaaaa gactcacgtt 1860

tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc 1920

gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca 1980

gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc 2040

agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact 2100

cctgatgatg catggttact caccactgcg atccccggca aaacagcatt ccaggtatta 2160

gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg 2220

ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct 2280

caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt 2340

aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg 2400

gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa 2460

ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc 2520

atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa 2580

tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt 2640

ttctaatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt 2700

ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc 2760

gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca 2820

atcgtatgtg aatgctggtc gctatactgc tgtcgattcg atacttgaat cgtctccccc 2880

ggtccgtttg ttctatactt ctctctgcta tacctacaag caaggtaatc ggaagtagta 2940

ttacgcagga atatcccgcg cgaagctaca atttttggac tccaacgtca aagcagggga 3000

gtcagaagtc ccctctaaaa ttgcctatcg tcatgtaatt agttatgtca cgcttacatt 3060

cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct 3120

aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa 3180

tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc 3240

ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa cgacggtaga cgccaactac 3300

gctgacagac cgatttgttt aagattagaa gatttttagc cgcgccgcaa tcggaaccag 3360

caaactcaat tctgggaaca gtttaaaata ctagtaatta cgatagccga gaaacggact 3420

aagtccgcga gacgtctaga gttccatccc aatggcgcgc cgagcttggc tcgagcatgg 3480

tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 3540

ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 3600

ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 3660

ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 3720

gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 3780

atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 3840

caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 3900

cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 3960

taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 4020

ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 4080

tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 4140

gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 4200

ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 4260

aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 4320

agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 4380

agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 4440

cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 4500

gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 4560

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 4620

gagtaaactt ggtctgacag ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta 4680

ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa 4740

aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact 4800

cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag 4860

aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc 4920

cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa 4980

ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga 5040

caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata 5100

ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc agggatcgca 5160

gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc 5220

ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta 5280

cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt 5340

gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc 5400

atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcatactc 5460

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 5520

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 5580

ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 5640

acgaggccct ttcgtc 5656

<210> 13

<211> 26

<212> DNA

<213> Synthesis

<400> 13

ggatgatgaa ctactttccc tcactg 26

<210> 14

<211> 26

<212> DNA

<213> Synthesis

<400> 14

aggcaatttt agaggggact tctgac 26

<210> 15

<211> 4438

<212> DNA

<213> Synthesis

<400> 15

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatggaac 420

ggatcccgtc tcaaacgacg gtagacgcca actacgctga cagaccgatt tgtttaagat 480

tagaagattt ttagccgcgc cgcaatcgga accagcaaac tcaattctgg gaacagttta 540

aaatactagt aattacgata gccgagaaac ggactaagtc cgcggtaccg ctatcaaaaa 600

cgatagatcg attaggatga ctttgaaatg actccgcagt ggactggccg ttaatttcaa 660

gcgtgagtaa aatagtgcat gacaaaagat gagctaggct tttgtaaaaa tatcttacgt 720

tgtaaaattt tagaaatcat tatttccttc atatcatttt gtcattgacc ttcagaagaa 780

aagagccgac caataatata aataaataaa taaaaataat attccattat ttctaaacag 840

attcaatact cattaaaaaa ctatatcaat taatttgaat taacaggtga caaagcgcca 900

aggaactgta atatatagct acgccctatc tggacgattg ggcgactttt acgtacggtt 960

gctcaattcc tacgcaactt aatatatttt gcaacggtta aatcggcttg aagctcgggc 1020

tatccaactc gcggactaga atccatgtat ttacctttgc cattgaatgt atcaagaacg 1080

ttaatcagat tttctttgct ctgtgctaga ggttctttgg aatataccaa aggtggtgcg 1140

ttattcttag ataataaaaa tcttctcctc gacaccatca aagatatcga aaacgatagg 1200

cttttggatt tacctggaat tgcctcttgg cacacgctga aactgtttga catgagcatc 1260

aacctgttgt tgaaagcacc taatgttaag gttgaaagac tggataaatt cttggaaaag 1320

aaattgaatt actacaatag attgatgggg ttaccaccgg ccacaaccac atccttaaaa 1380

ccattatttg gctctcaatc gaagaacagc ctggaaaata gacaaagaac acctaatgtc 1440

aaaagagaaa acccagaaca cgagtatctt tatggaaacg atagtaataa taacaataat 1500

tctgaagcgg gtcactctcc aatgacaaat acaactaatg gtaataagag attaaagtat 1560

gaaaaagatg caaaacgaaa tgcaaaagat ggcggtatat ccaaggggga gaacgcacat 1620

aatttccaga atgataccaa aaaaaacatg tccaccagca atctgtttcc attttcgttt 1680

agtaatacag accttactgc gctgttcacc catcctgaag gaccgaattg tacgaatact 1740

aataatggca tcggcgattt aatctctaat tattagttaa agttttataa gcatttttat 1800

gtaacgaaaa ataaattggt tcatattatt actgcactgt cacttaccat ggaaagacca 1860

gacaagaagt tgccgacagt ctgttgaatt ggcctggtta ggcttaagtc tgggtccgct 1920

tctttacaaa tttggagaat ttctcttaaa cgatatgtat attcttttcg ttggaaaaga 1980

tgtcttccaa aaaaaaaacc gatgaattag tggaaccaag gaaaaaaaaa gaggtatcct 2040

tgattaagga acagagctcc cagacgatac agaggctaag aataacgcag ataatcgctc 2100

taacgaaacg tactaaaaga tttcttttga agtaactaga taccctggtc ttatactagg 2160

tatctttgtc agaaacggcc taagactaca gtaagagcag ttggaaccta gagacgtcta 2220

gagttccatc ccaatggcgc gccgagcttg gctcgagcat ggtcatagct gtttcctgtg 2280

tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 2340

gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 2400

ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 2460

ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 2520

gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 2580

tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 2640

aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 2700

aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 2760

ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 2820

tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 2880

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 2940

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 3000

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 3060

acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 3120

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 3180

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 3240

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 3300

aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 3360

ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 3420

agttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca 3480

ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc 3540

cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa 3600

cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg 3660

actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc 3720

cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat 3780

tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc 3840

gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga 3900

tattcttcta atacctggaa tgctgttttc ccagggatcg cagtggtgag taaccatgca 3960

tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag 4020

tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga 4080

aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg 4140

acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc 4200

ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat 4260

tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 4320

aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 4380

accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 4438

<210> 16

<211> 36

<212> DNA

<213> Synthesis

<400> 16

gacaaagcgc caaggaactg taatatatag ctacgc 36

<210> 17

<211> 30

<212> DNA

<213> Synthesis

<400> 17

gccattatta gtattcgtac aattcggtcc 30

<210> 18

<211> 35

<212> DNA

<213> Synthesis

<400> 18

accaggtctc aaggtatgtc cgtttccaag attgc 35

<210> 19

<211> 39

<212> DNA

<213> Synthesis

<400> 19

accaggtctc acgatttata acaataaagc agctgcacc 39

<210> 20

<211> 38

<212> DNA

<213> Synthesis

<400> 20

accaggtctc aaggtatgtc tagcgaagat aagaaacc 38

<210> 21

<211> 41

<212> DNA

<213> Synthesis

<400> 21

accaggtctc acgatctaag cctttttgtt gatttcttga g 41

<210> 22

<211> 37

<212> DNA

<213> Synthesis

<400> 22

accaggtctc aaggtatgtt gtctaacgct aagctcc 37

<210> 23

<211> 39

<212> DNA

<213> Synthesis

<400> 23

accaggtctc acgattcata aaagcatcat aactgcacc 39

<210> 24

<211> 36

<212> DNA

<213> Synthesis

<400> 24

accaggtctc aaggtatgca aagcccatat ccaatg 36

<210> 25

<211> 34

<212> DNA

<213> Synthesis

<400> 25

accaggtctc acgattcagt ccatgtgtgg gaag 34

<210> 26

<211> 59

<212> DNA

<213> Synthesis

<400> 26

accaggtctc aaggtatggt atggtatgat cataacacgc attctgaaaa tgttatctg 59

<210> 27

<211> 59

<212> DNA

<213> Synthesis

<400> 27

accaggtctc acgatttaag atggtgtatt atgtcgcctt ttgcaatact ttaatttct 59

<210> 28

<211> 35

<212> DNA

<213> Synthesis

<400> 28

accaggtctc aaggtatgtg tgaatacagc aaggc 35

<210> 29

<211> 40

<212> DNA

<213> Synthesis

<400> 29

accaggtctc acgatttaaa acccatgttg actcatgatc 40

<210> 30

<211> 52

<212> DNA

<213> Synthesis

<400> 30

accaggtctc aaggtatgga aatcaagcca gttgaggtta ttgatggcgt tc 52

<210> 31

<211> 59

<212> DNA

<213> Synthesis

<400> 31

accaggtctc acgatctatg attcagctaa tttagtattt tccatttctt gagtgcatg 59

<210> 32

<211> 55

<212> DNA

<213> Synthesis

<400> 32

accaggtctc aaggtatggc taacgtagaa aaaccaaacg attgttcagg ctttc 55

<210> 33

<211> 59

<212> DNA

<213> Synthesis

<400> 33

accaggtctc acgattcaac tgtttccctt tagatgattt tccaaagtgt ggaaatcac 59

<210> 34

<211> 58

<212> DNA

<213> Synthesis

<400> 34

accaggtctc aaggtatgtc acaggtttgg cataattcga attcgcaatc aaacgatg 58

<210> 35

<211> 59

<212> DNA

<213> Synthesis

<400> 35

accaggtctc acgatttatt gaatcttagc ccccttgtct gaagattcag tattcccag 59

<210> 36

<211> 45

<212> DNA

<213> Synthesis

<400> 36

accaggtctc aaggtatgtc tgctgctcct gtccaagaca aagac 45

<210> 37

<211> 55

<212> DNA

<213> Synthesis

<400> 37

accaggtctc acgattcaag tagcacttgt ccaattattc caatcccata cattg 55

<210> 38

<211> 57

<212> DNA

<213> Synthesis

<400> 38

accaggtctc aaggtatgaa gctactgtct tctatcgaac aagcatgcga tatttgc 57

<210> 39

<211> 55

<212> DNA

<213> Synthesis

<400> 39

accaggtctc acgatttact ctttttttgg gtttggtggg gtatcttcat catcg 55

<210> 40

<211> 42

<212> DNA

<213> Synthesis

<400> 40

accaggtctc aaggtatgac tacagatcct tctgtcaaat tg 42

<210> 41

<211> 41

<212> DNA

<213> Synthesis

<400> 41

accaggtctc acgattcaaa tagaagagtt ggatttgtcc t 41

<210> 42

<211> 41

<212> DNA

<213> Synthesis

<400> 42

accaggtctc aaggtatggg tgtcatcaag aagaaaagat c 41

<210> 43

<211> 48

<212> DNA

<213> Synthesis

<400> 43

accaggtctc acgatttaaa aaaatacagc atcaagatct ccaaattc 48

<210> 44

<211> 52

<212> DNA

<213> Synthesis

<400> 44

accaggtctc aaggtatgaa cataccacag cgtcaattta gcaacgaaga gg 52

<210> 45

<211> 46

<212> DNA

<213> Synthesis

<400> 45

accaggtctc acgatctata cactcgcttc tgtcatgctc gagtcc 46

<210> 46

<211> 41

<212> DNA

<213> Synthesis

<400> 46

accaggtctc aaggtatgct agtcttcgga cctaatagta g 41

<210> 47

<211> 39

<212> DNA

<213> Synthesis

<400> 47

accaggtctc acgattcaaa aatcaccgtg ctttttgtg 39

<210> 48

<211> 35

<212> DNA

<213> Synthesis

<400> 48

accaggtctc aaggtatgtc cggtagaggt aaagg 35

<210> 49

<211> 38

<212> DNA

<213> Synthesis

<400> 49

accaggtctc acgatttaac caccgaaacc gtataagg 38

<210> 50

<211> 40

<212> DNA

<213> Synthesis

<400> 50

accaggtctc aaggtatgtc attcgacgac ttacacaaag 40

<210> 51

<211> 40

<212> DNA

<213> Synthesis

<400> 51

accaggtctc aaggtatgtc attcgacgac ttacacaaag 40

<210> 52

<211> 34

<212> DNA

<213> Synthesis

<400> 52

accaggtctc aaggtatgac caccactgcc caag 34

<210> 53

<211> 34

<212> DNA

<213> Synthesis

<400> 53

accaggtctc acgattcaaa cctttgcgcc ggtg 34

<210> 54

<211> 40

<212> DNA

<213> Synthesis

<400> 54

accaggtctc aaggtatggc tggtgaaact tttgaatttc 40

<210> 55

<211> 40

<212> DNA

<213> Synthesis

<400> 55

accaggtctc acgatttaat caacttcttc catctcggtg 40

<210> 56

<211> 42

<212> DNA

<213> Synthesis

<400> 56

accaggtctc aaggtatgtc ccgtgaccta caaaaccatt tg 42

<210> 57

<211> 42

<212> DNA

<213> Synthesis

<400> 57

accaggtctc acgatttaac tgtcatcagc atatgggcca tc 42

<210> 58

<211> 42

<212> DNA

<213> Synthesis

<400> 58

accaggtctc aaggtatgaa gttccaagtt gttttatctg cc 42

<210> 59

<211> 38

<212> DNA

<213> Synthesis

<400> 59

accaggtctc acgattcatg ggaaaatgct ttccagag 38

<210> 60

<211> 45

<212> DNA

<213> Synthesis

<400> 60

accaggtctc aaggtatgtc tgctgttaac gttgcacctg aattg 45

<210> 61

<211> 49

<212> DNA

<213> Synthesis

<400> 61

accaggtctc acgatttaac caatcaactc accaaacaaa aatggggtg 49

<210> 62

<211> 34

<212> DNA

<213> Synthesis

<400> 62

accaggtctc aaggtatgca attctctacc gtcg 34

<210> 63

<211> 36

<212> DNA

<213> Synthesis

<400> 63

accaggtctc acgatttaca acaataaagc ggcagc 36

<210> 64

<211> 41

<212> DNA

<213> Synthesis

<400> 64

accaggtctc aaggtatgca attacattca cttatcgctt c 41

<210> 65

<211> 43

<212> DNA

<213> Synthesis

<400> 65

accaggtctc acgatttaca ttatagacat gatgattgcc gtc 43

<210> 66

<211> 50

<212> DNA

<213> Synthesis

<400> 66

accaggtctc aaggtatgtt taagtctgtt gtttattcgg ttctagccgc 50

<210> 67

<211> 56

<212> DNA

<213> Synthesis

<400> 67

accaggtctc acgatttatt gttttaatag ggtatcgttg tagtgagtag tattcc 56

<210> 68

<211> 35

<212> DNA

<213> Synthesis

<400> 68

accaggtctc aaggtatggg ctcaaaagta gcagg 35

<210> 69

<211> 49

<212> DNA

<213> Synthesis

<400> 69

accaggtctc acgattcagc ttgtatctga gaattttctt ttcttattc 49

Claims (8)

1. A method for constructing a fermentation strain, comprising deleting a Saccharomyces cerevisiae strain from the genomeCAT8Genes and in the genome of the Saccharomyces cerevisiae strainCAT8For integration site, the target gene is integrated into the deletionCAT8The genome of the Saccharomyces cerevisiae strain after the gene; the target gene isTIP1；

Wherein the saccharomyces cerevisiae strain is named as CE10, and the preservation number of the saccharomyces cerevisiae strain is CGMCC NO.24860; the saidTIP1The sequence of (2) is shown as SEQ ID NO. 2.

2. The method for constructing a fermentation strain according to claim 1, wherein the target gene is integrated into the deletionCAT8The steps in the genome of the Saccharomyces cerevisiae strain after the gene comprise: integration of a linear fragment containing the gene of interest into a deletionCAT8And (3) in the genome of the saccharomyces cerevisiae strain after the gene, and over-expressing the target gene.

3. A fermentation strain produced by the method of constructing a fermentation strain according to claim 1 or 2.

4. A method for producing bioethanol comprising the fermentative cultivation of the strain according to claim 3 in a culture system comprising xylose.

5. The method for producing bioethanol according to claim 4, characterized in that the concentration of xylose in the culture system is 30-50g/L.

6. The method for producing bioethanol according to claim 4, characterized in that glucose is further contained in the culture system.

7. The method for producing bioethanol according to claim 6, characterized in that the concentration of glucose in the culture system is 70-100g/L.

8. The method for producing bioethanol according to claim 4, wherein the fermentation culture temperature is 30-35 ℃.

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