CN115232757A - Saccharomyces cerevisiae strain, fermentation strain, construction method of fermentation 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 of the saccharomyces cerevisiae strain 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 increased; can also improve the tolerance to acid inhibitors such as acetic acid, and further improve the yield of the bioethanol.

Description

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

Background

The bioethanol is obtained by using renewable biomass as a raw material through a series of biotransformation processes, 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, the bioethanol has higher evaporation enthalpy, laminar flame speed and heat of vaporization; in addition, the bioethanol has high oxygen content, is beneficial to improving combustion efficiency, has low sulfur content, and is beneficial to reducing the emission of sulfur dioxide and other air pollutants.

Currently, bioethanol is mainly obtained from lignocellulose-rich feedstocks, such as crop residues (crop straws, sugar cane bagasse, corn cobs, etc.), energy crops (perennial herbaceous plants such as miscanthus or switchgrass, etc.), aquatic plants (water hyacinths, etc.), forest materials, and municipal solid biomass, etc., in abundant reserves, renewable, and relatively inexpensive.

Ethanol produced by lignocellulose biomass is subjected to four steps of pretreatment, hydrolysis, fermentation and distillation. Among them, the pretreatment process is to remove lignin and hemicellulose from lignocellulose matrix, reduce the crystallinity of cellulose, increase the porosity and surface area of biomass, and is a key step for converting biomass raw material into fermentable sugar and biofuel. Fermentation is a process of converting soluble sugars 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 large saccharide in the lignocellulose hydrolysate, and the realization of the efficient and rapid conversion of the xylose is an economic means for producing bioethanol by utilizing the lignocellulose biomass.

However, during the pretreatment of the lignocellulose biomass, acid inhibitors such as acetic acid can be formed, the rate and the yield of the wild saccharomyces cerevisiae during the subsequent fermentation can be influenced, and the wild saccharomyces cerevisiae can only utilize glucose to metabolize but cannot utilize xylose to metabolize, so that the yield of bioethanol produced by the wild saccharomyces cerevisiae is low, and the large-scale production of bioethanol is not facilitated.

Disclosure of Invention

The application aims to provide a saccharomyces cerevisiae strain, a fermentation strain, a construction method of the saccharomyces cerevisiae strain and a bioethanol production method, and aims 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 application provides a saccharomyces cerevisiae strain, named as CE10, with a accession number of CGMCC No.24860.

Biological material: CE10, category name: saccharomyces cerevisiae, deposited at CGMCC (China general microbiological culture Collection center) on 09.05.2022 with the addresses of: the microbial research institute of China academy of sciences No.3, xilu No.1, beijing, chaoyang, with the collection number of CGMCC NO.24860.

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

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

The target genes MSN2, TIP1, YRB1 or SPI1 are respectively integrated into the genome of the CE10 saccharomyces cerevisiae strain to construct a fermentation strain, the constructed fermentation strain can effectively utilize xylose for metabolism compared with wild saccharomyces cerevisiae, and the utilization efficiency of the strain on xylose and the tolerance of the strain on acid inhibitors such as acetic acid and the like can be further effectively improved compared with the CE10 saccharomyces cerevisiae strain, so that 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 then 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 sequence of MSN2 is shown as SEQ ID NO.1, the sequence of TIPI is shown as SEQ ID NO.2, the sequence of YRB1 is shown as SEQ ID NO.3, and the sequence of SPII is shown as SEQ ID NO. 4.

The CAT8 gene in the genome of the saccharomyces cerevisiae strain CE10 is deleted, so that metabolic pathways of the subsequently prepared fermentation strain during fermentation flow more in the direction of ethanol fermentation, and the yield of bioethanol is improved. And integrating the MSN2, TIP1, YRB1 or SPI1 target genes into the genome of the saccharomyces cerevisiae strain without the CAT8 gene by taking CAT8 as an integration site to construct a fermentation strain, wherein the constructed fermentation strain can obviously improve the utilization efficiency of the strain on xylose and the tolerance of the strain on acid inhibitors such as acetic acid and the like, and further improve the yield of bioethanol.

In one possible embodiment, the step of integrating the gene of interest into the genome of the s.cerevisiae strain after deletion of the CAT8 gene comprises: and integrating a linear fragment containing the target gene into the genome of the saccharomyces cerevisiae strain after the CAT8 gene is deleted, and enabling the target gene to be over-expressed.

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

In a fourth aspect, the present application provides a fermentation strain, which is produced by the method for constructing the fermentation strain provided in the second or third aspect.

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 on xylose and the tolerance of the strain on 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 culture 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 by the first aspect of the present application and the fermentation strain provided by the fourth aspect of the present application can effectively utilize xylose for metabolism, thereby increasing the yield of bioethanol; the CE10 saccharomyces cerevisiae strain and the fermentation strain provided by the fourth aspect of the application are adopted to carry out 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 30-50g/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 capable of well metabolizing xylose, and further more bioethanol can be generated. 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 by the fourth aspect of the application can perform xylose metabolism and glucose metabolism, and further more bioethanol can be generated.

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

The concentration of glucose in the culture system is 70-100g/L, which can ensure that the strain provided by the application can well utilize glucose to metabolize, and is further favorable for producing more bioethanol.

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

Drawings

To more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

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 production after 36h and 48h fermentation of the strains provided in examples 1-5 of the present application, respectively.

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

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

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

Biological material deposit description:

biological material: CE10, category name: saccharomyces cerevisiae, deposited at CGMCC (China general microbiological culture Collection center) on 09.05.2022 with the addresses of: the microbial research institute of China academy of sciences, no.3 of Xilu No.1 of Beijing, chaoyang, with the collection number of CGMCC NO.24860.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The saccharomyces cerevisiae strain, the fermentation strain, the construction method and the application thereof, and the method for producing bioethanol provided by the present application are specifically described below.

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

Biological material: CE10, category name: saccharomyces cerevisiae, deposited at CGMCC (China general microbiological culture Collection center) on 09.05.2022 with the addresses of: the microbial research institute of China academy of sciences No.3, xilu No.1, beijing, chaoyang, with the collection number of CGMCC NO.24860.

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; can also improve the tolerance of the strain to acid inhibitors such as acetic acid and the like, and further improve the yield of the bioethanol.

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

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

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

Respectively integrating a target gene MSN2 (transcription factor), TIP1 (model gene), YRB1 (model gene) or SPI1 (cell wall gene) 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, so that the yield of bioethanol is increased, 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 deleting the CAT8 gene in the genome of the CE10 saccharomyces cerevisiae strain, and then integrating the target gene into the genome of the CE10 saccharomyces cerevisiae strain after the CAT8 gene is deleted by taking 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 sequence of MSN2 is shown as SEQ ID NO.1, the sequence of TIPI is shown as SEQ ID NO.2, the sequence of YRB1 is shown as SEQ ID NO.3, and the sequence of SPII is shown as SEQ ID NO. 4.

The CAT8 gene in the genome of the saccharomyces cerevisiae strain CE10 is deleted, so that metabolic pathways of the subsequently prepared fermentation strain during fermentation flow more in the direction of ethanol fermentation, and the yield of bioethanol is improved. And integrating the MSN2, TIP1, YRB1 or SPI1 target genes into the genome of the saccharomyces cerevisiae strain without the CAT8 gene by taking CAT8 as an integration site to construct a fermentation strain, wherein the constructed fermentation strain can obviously improve the utilization efficiency of the strain on xylose and the tolerance of the strain on acid inhibitors such as acetic acid and the like, and further improve the yield of bioethanol.

Further, the step of integrating the gene of interest into the genome of the s.cerevisiae strain after deletion of the CAT8 gene comprises: and integrating a linear fragment containing the target gene into the genome of the saccharomyces cerevisiae strain after the CAT8 gene is deleted, and enabling the target gene to be over-expressed.

CAT8 is used as an integration site and the target gene is over-expressed, which is beneficial to constructing a stable fermentation strain.

In the examples of the present application, the method for preparing a linear fragment containing a gene of interest comprises: firstly, the carrier fragment and the target gene fragment are connected by enzyme digestion by a Golden Gate Assembly (GGA) method so as to construct a plasmid containing the target gene. The plasmid containing the target gene was electrically transferred to Escherichia coli competence, and colonies of positive clones were selected on a solid LB medium with kanamycin resistance. And selecting a certain number of positive clone colonies, confirming whether a correct band is amplified or not through colony PCR, and extracting corresponding plasmids if the band size and the sequencing result are correct. And then using the extracted plasmid as a template to amplify a linear fragment containing the target gene.

In the present examples, 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 gene of interest and two additional linear fragments having homologous fragments to the linear fragment containing the gene of interest were integrated together by lithium acetate transformation into the CAT8 site of the CE10 s.cerevisiae strain.

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 on xylose and the tolerance of the strain on acid inhibitors such as acetic acid can be further effectively improved, and the yield of bioethanol is further improved.

In the application, when a fermentation strain is constructed by adopting a method of firstly deleting CAT8 gene in the genome of a CE10 saccharomyces cerevisiae strain, then using CAT8 gene in the genome of the CE10 saccharomyces cerevisiae strain as an integration site, integrating a target gene-containing fragment into the genome of the CE10 saccharomyces cerevisiae strain after the CAT8 gene is deleted by utilizing the principle of homologous recombination, and enabling the target gene to be over-expressed, when the target gene is MSN2, the fermentation strain is named as WXY246; when the target gene is TIP1, the fermentation strain is named as WXY247; when the gene of interest is YRB1, the fermentation strain is named WXY249; when the target gene is SPI1, the fermentation strain is named as 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 increased, 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 strain can further improve the utilization efficiency of xylose, the tolerance to acid inhibitors such as acetic acid and the like and the yield of bioethanol.

Wherein, the culture conditions of the WXY246 fermentation strain are as follows: a single colony of WXY 246-fermenting strain was picked up and cultured in YPD medium at 30 ℃ and 200rpm with shaking.

The storage conditions of the WXY246 fermentation strain are as follows: inoculating WXY246 zymocyte into YPD solid plate culture medium, culturing at 30 deg.C for 48-72 hr, and short-term preserving at 4 deg.C. Or inoculating the cultured WXY246 zymocyte liquid into an 80% (v/v) sterile glycerol freezing storage tube, and freezing for long-term storage at-80 ℃.

Wherein, the culture conditions of the WXY247 fermentation strain are as follows: single colony of WXY247 fermenting strain was picked up and cultured in YPD medium at 30 ℃ and 200rpm with shaking.

The preservation conditions of the WXY247 fermentation strain are as follows: inoculating WXY247 zymocyte into YPD solid plate culture medium, culturing at 30 deg.C for 48-72 hr, and short-term storing at 4 deg.C. Or inoculating the cultured WXY247 zymocyte liquid into an 80% (v/v) sterile glycerol freezing tube, and freezing at-80 deg.C for long-term storage.

Wherein, the culture conditions of the WXY249 fermentation strain are as follows: a single colony of WXY 249-fermenting strain was picked up and cultured in YPD medium at 30 ℃ and 200rpm with shaking.

The storage conditions of the WXY249 fermentation strain are: inoculating WXY249 zymocyte into YPD solid plate culture medium, culturing at 30 deg.C for 48-72 hr, and short-term storing at 4 deg.C. Or inoculating the cultured WXY249 fermenting bacteria liquid into 80% (v/v) sterile glycerin freezing tube for long-term preservation at-80 deg.C.

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

The preservation conditions of the WXY256 fermentation strain are as follows: inoculating WXY256 zymocyte into YPD solid plate culture medium, culturing at 30 deg.C for 48-72 hr, and short-term preserving at 4 deg.C. Or inoculating the cultured WXY256 zymocyte liquid into an 80% (v/v) sterile glycerol freezing storage tube, and freezing for long-term storage at-80 ℃.

The present application also provides a method for the biological production of bioethanol, comprising the fermentative culture of the provided CE10 saccharomyces cerevisiae strain and the fermentation strain in a culture system comprising 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 method can be used for large-scale ethanol production.

Furthermore, 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 capable of well metabolizing xylose, and further more bioethanol can be generated. Illustratively, the concentration of xylose in the culture system can be 30g/L, 35g/L, 40g/L, 45g/L, or 50g/L, and so forth.

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 perform xylose metabolism and glucose metabolism, and further more bioethanol can be generated.

Furthermore, the concentration of glucose in the culture system is 70-100g/L, which can ensure that the strain provided by the application can well utilize glucose to metabolize, and is further beneficial to producing more bioethanol. Illustratively, the concentration of glucose in the culture system can be 70g/L, 75g/L, 80g/L, 90g/L, or 100g/L, and so forth.

Furthermore, 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 method is favorable for better metabolic production of bioethanol. Illustratively, the temperature of the fermentation culture may be 30 ℃, 32 ℃, or 35 ℃ and the like.

Example 1

This example provides a strain of CE10 s.cerevisiae (accession number CGMCC NO. 24860).

Example 2

This example provides a WXY246 fermentation strain, constructed using the following steps:

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

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

Firstly, determining whether the size of the band of the amplified MSN2 target gene fragment is correct through agarose gel electrophoresis, and then comparing the base sequence of the amplified MSN2 target gene fragment through sequencing. If the size of the band and the gene sequence are correct, the obtained T1-0 carrier segment and the MSN2 target gene segment are subjected to enzyme digestion and connection by a GGA method to construct a plasmid T1-0-MSN2. Wherein, in the process of enzyme digestion and connection, the used endonuclease is Bsa I-HFv 2, and the used Ligase is T4 DNA Ligase.

The plasmid T1-0-MSN2 was electrically transferred to E.coli competence, and colonies of positive clones were selected on solid LB medium with kanamycin resistance. And selecting a certain number of positive clone colonies, confirming whether a correct band is amplified or not through colony PCR, and determining that the T1-0-MSN2 plasmid can be extracted if the band size and the sequencing result are correct. And amplifying an L1-pPGK1-MSN2-tPGI1-L2 linear fragment by taking the extracted T1-0-MSN2 plasmid as a template and taking L1-F with a sequence shown as SEQ ID NO.10 and L2-R with a sequence shown as SEQ ID NO.11 as upstream and downstream primers respectively. Wherein the step of electrically transforming the plasmid T1-0-MSN2 to the escherichia coli competence comprises: cleaning the electric rotating cup with 75% alcohol, reversing the electric rotating cup on dust-free paper, air drying, and pre-cooling the electric rotating cup on ice coated with plastic film for 20min. The electroporation competent cells were removed from the-80 ℃ freezer and inserted into ice to be thawed. Sucking 2.5 mu L of the ligation product T1-0-MSN2, adding the ligation product into 50 mu L of electrotransformation escherichia coli competence, uniformly mixing, adding a uniformly mixed sample into the aperture of an electrotransfer cup, and inserting the sample into an electrotransfer instrument for pulse electric shock. To the shocked sample was added 200. Mu.L of nonreactive LB, and the mixture was blown and mixed well and transferred to a 1.5mL centrifuge tube. Culturing the electro-transferred bacterial liquid in a shaker at 37 ℃ and 200rpm for 1h, centrifuging at 12000rpm for 2min, collecting thalli, leaving 50 mu L of liquid, uniformly mixing, coating on a corresponding resistant plate, and performing inverted culture in an incubator at 37 ℃ for 16h.

(2) CAT8UP-G418-L1 and L2-CAT8DOWN, in which a fragment homologous to the linear fragment containing the gene of interest was present, were amplified.

T2-CAT8UP-G418-L1 with the sequence shown in SEQ ID NO.12 is taken as a template, CAT8UP-F with the sequence shown in SEQ ID NO.13 and L1-R with the sequence shown in SEQ ID NO.14 are taken as an upstream primer and a downstream primer respectively, and a linear fragment CAT8UP-G418-L1 is amplified.

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

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

The CAT8 gene of the genome of the CE10 Saccharomyces cerevisiae strain is deleted, and the linear fragments containing the target gene, L1-pPGK1-MSN 2-tPGGI 1-L2, CAT8UP-G418-L1 and L2-CAT8DOWN, are transformed into the CE10 Saccharomyces cerevisiae strain by a Saccharomyces cerevisiae lithium acetate transformation method to over-express the MSN2 gene, with CAT8 as an integration site. Wherein, the method for converting the saccharomyces cerevisiae lithium acetate comprises the following steps: 20mL of YPD liquid medium and a single colony of a CE10 Saccharomyces cerevisiae strain were added to a conical flask, and cultured at 30 ℃ for 12 hours in a shaker at 200rpm to obtain a bacterial solution. Adding bacteria solution into liquid culture medium containing 20mLYPD, and adding OD ₆₀₀ Adjusting to 0.2, culturing at 30 deg.C and 200rpm for 4 hr to make OD of cultured strain liquid ₆₀₀ Is 0.7. Add 1mLOD to 1.5mL centrifuge tube ₆₀₀ The cells were centrifuged at 3000g for 2min at 25 ℃ for 0.7 of the bacterial solution, and the supernatant was discarded. Suction 1mLddH ₂ Washing the bacterial liquid with water, centrifuging at 3000gFor 2min, discard the supernatant. Then suck 1mlddH ₂ Washing the bacterial solution with water, centrifuging for 2min at 3000g, and discarding the supernatant. The washed cells were resuspended in 100. Mu.L of 0.1MLiAc solution, cultured at 30 ℃ for 10min in an incubator, centrifuged at 3000g for 2min, and the supernatant was discarded. Meanwhile, the ssDNA solution was placed on a heating block at 100 ℃ and heated for 10min, and immediately after heating, the ssDNA solution was inserted into ice. The cells were resuspended in 20. Mu.L of ddH2O, and then 360. Mu.L of 50% PEG solution, 55. Mu.L of 1MLiAc solution, 75. Mu.L of the treated ssDNA solution and 1500ng of L1-pPGK1-MSN2-tPGI1-L2, 1500ng of CAT8UP-G418-L1 and 1500ng of L2-CAT8 DOWNdH were added in that order ₂ And 40 mu.L of O mixed solution. The mixture was vortexed vigorously at 1400rpm until it was completely mixed, incubated in an incubator at 30 ℃ for 30min, and then water-bathed in a 42 ℃ water bath for 30min. After the completion of the water bath, 7500g of the cells were centrifuged for 2min, and the centrifuged cells were suspended in 1mLYPD liquid medium and cultured for 3 hours at 30 ℃ in a shaker at 200 rpm. Centrifuge at 3000g for 2min and discard the supernatant. Add 1mL of ddH ₂ The cells were washed with water O, centrifuged at 3000g for 2min and the supernatant discarded. Add 1mL of ddH ₂ The cells were washed with water O, centrifuged at 3000g for 2min and the supernatant discarded. 50 μ L of the resuspended cells were spread on a resistant solid medium and cultured in an inverted state in an incubator at 30 ℃ for 4 days.

Example 3

This example provides a WXY247 fermentation strain, and the construction method of the WXY247 fermentation strain is basically similar to that of example 2, except that: the target gene in the embodiment is TIP1, and the amplification of the target gene segment of TIP1 adopts the following steps: a crude extracted wild saccharomyces cerevisiae genome 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 an upstream primer and a downstream primer, and a TIP1 target gene segment with a sequence shown as SEQ ID NO.2 is amplified through PCR.

Example 4

This example provides a WXY249 fermentation strain, which was constructed substantially similarly to example 2, except that: the target gene in this example is YRB1, and the amplification of the target gene fragment of YRB1 adopts the following steps: taking crude wild type saccharomyces cerevisiae genome as a template, taking YRB1-gga-F with a sequence shown as SEQ ID NO.20 and YRB1-gga-R with a sequence shown as SEQ ID NO.21 as upstream and downstream primers respectively, and amplifying a YRB1 target gene segment with a sequence shown as SEQ ID NO.3 by PCR.

Example 5

The embodiment provides a WXY256 fermentation strain (the construction method of the WXY256 fermentation strain is basically similar to that of embodiment 2, and the difference is that the target gene in the embodiment is SPI1, and the amplification of the SPI1 target gene fragment adopts the following steps that crude wild saccharomyces cerevisiae genome is taken as a template, SPI1-gga-F with the sequence shown as SEQ ID NO.22 and SPI1-gga-R with the sequence shown as SEQ ID NO.23 are respectively taken as upstream and downstream primers, and the SPI1 target gene fragment with the sequence shown as SEQ ID NO.4 is amplified through PCR.

Comparative examples 1 to 23

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

TABLE 1

Experimental example 1

When the strains provided in examples 1 to 5 were subjected to a mixed sugar fermentation experiment, the results of xylose utilization after 48h fermentation of the strains are shown in fig. 1, the results of ethanol production after 36h and 48h fermentation of the strains are shown in fig. 2, and the results of acetic acid consumption after 48h fermentation of the strains are shown in fig. 3. Wherein, the steps of the fermentation experiment of the mixed sugar are as follows: adding the cultured bacterial liquid into a fermentation culture medium, adjusting OD600 to 1, and culturing in a shaking table at 30 ℃ and 200rpm for 48h in a dark place. 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 And O, obtaining a fermentation medium.

As can be seen from FIG. 1, the strains of examples 1 to 5 were all able to metabolize xylose efficiently; since the wild-type Saccharomyces cerevisiae cannot utilize xylose for metabolism, it is shown that the strains provided in examples 1-5 can significantly improve the utilization efficiency of xylose compared to the wild-type Saccharomyces cerevisiae. And the xylose utilization efficiency of the strains provided in examples 2-4 after 48h fermentation is higher than that of the strain provided in example 1 after 48h fermentation, which shows that the integration of MSN2, TIP1, YRB1 or SPI1 into the genome of the CE10 Saccharomyces cerevisiae strain can further improve the xylose utilization efficiency of the strains.

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

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

Experimental example 2

The results of ethanol production after 36h and 48h fermentation of the strains provided in example 2 and comparative examples 1-12 by mixed sugar fermentation experiments are shown in FIG. 4. The fermentation test with mixed sugar was the same as in example 1.

The target genes of example 2 and comparative examples 1-12 are transcription factors, and it can be seen from FIG. 4 that the ethanol yield of the strain of example 2 after 36h and 48h fermentation is significantly higher than that of the strain of comparative examples 1-12 after 36h and 48h fermentation, which shows that the MSN2 transcription factor selected in example 2 is integrated into the genome of the CE10 Saccharomyces cerevisiae strain to effectively increase the bioethanol yield compared with the transcription factors adopted in comparative examples 1-12.

Experimental example 3

The results of ethanol production after 36h and 48h fermentation of the strains provided in examples 3-4 and comparative examples 13-17 by mixed sugar fermentation experiments are shown in FIG. 5. The fermentation test with mixed sugar was the same as in example 1.

The genes of interest of examples 3-4 and comparative examples 13-17 are the same expression pattern genes, and it can be seen from FIG. 5 that the ethanol yield of the strains of examples 3-4 after 36h and 48h of fermentation is significantly higher than that of the strains of comparative examples 13-17 after 36h and 48h of fermentation, which shows that the integration of the same expression pattern genes TIP1 and YRB1 selected in examples 3 and 4, respectively, into the genome of the CE10 s.cerevisiae strain can effectively increase the bioethanol yield compared to the same expression pattern genes adopted in comparative examples 13-17.

Experimental example 4

The results of ethanol production after 36h and 48h fermentation of the strains provided in example 5 and comparative examples 18-23 by mixed sugar fermentation experiments are shown in FIG. 6. The fermentation test with mixed sugar was the same as in example 1.

The target genes of example 5 and comparative examples 18-23 are cell wall genes, and it can be seen from fig. 6 that the ethanol yield of the strain of example 5 after 36h and 48h of fermentation is significantly higher than that of the strain of comparative examples 18-23 after 36h and 48h of fermentation, which shows that compared with the cell wall genes adopted in comparative examples 18-23, the cell wall gene SPI1 selected in example 5 is integrated into the genome of the CE10 saccharomyces cerevisiae strain to effectively increase the bioethanol yield.

In conclusion, compared with wild-type 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 present application can significantly improve xylose utilization efficiency and bioethanol yield, and thus has a good application prospect in the field of bioethanol production.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> university of capital 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> Artificial 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> Artificial 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> Artificial 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> Artificial 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> Artificial 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> Artificial Synthesis

<400> 6

accaggtctc aatcgaacaa atcgctctta aatatatacc taaag 45

<210> 7

<211> 53

<212> DNA

<213> Artificial Synthesis

<400> 7

accaggtctc aaccttgttt tatatttgtt gtaaaaagta gataattact tcc 53

<210> 8

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 8

accaggtctc aaggtatgac ggtcgaccat gatttc 36

<210> 9

<211> 46

<212> DNA

<213> Artificial Synthesis

<400> 9

accaggtctc acgatttaaa tgtctccatg ttttttatga gtcttg 46

<210> 10

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 10

cgtctccccc ggtccgtttg ttctatac 28

<210> 11

<211> 27

<212> DNA

<213> Artificial Synthesis

<400> 11

gcggacttag tccgtttctc ggctatc 27

<210> 12

<211> 5656

<212> DNA

<213> Artificial 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> Artificial Synthesis

<400> 13

ggatgatgaa ctactttccc tcactg 26

<210> 14

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 14

aggcaatttt agaggggact tctgac 26

<210> 15

<211> 4438

<212> DNA

<213> Artificial 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> Artificial Synthesis

<400> 16

gacaaagcgc caaggaactg taatatatag ctacgc 36

<210> 17

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 17

gccattatta gtattcgtac aattcggtcc 30

<210> 18

<211> 35

<212> DNA

<213> Artificial Synthesis

<400> 18

accaggtctc aaggtatgtc cgtttccaag attgc 35

<210> 19

<211> 39

<212> DNA

<213> Artificial Synthesis

<400> 19

accaggtctc acgatttata acaataaagc agctgcacc 39

<210> 20

<211> 38

<212> DNA

<213> Artificial Synthesis

<400> 20

accaggtctc aaggtatgtc tagcgaagat aagaaacc 38

<210> 21

<211> 41

<212> DNA

<213> Artificial Synthesis

<400> 21

accaggtctc acgatctaag cctttttgtt gatttcttga g 41

<210> 22

<211> 37

<212> DNA

<213> Artificial Synthesis

<400> 22

accaggtctc aaggtatgtt gtctaacgct aagctcc 37

<210> 23

<211> 39

<212> DNA

<213> Artificial Synthesis

<400> 23

accaggtctc acgattcata aaagcatcat aactgcacc 39

<210> 24

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 24

accaggtctc aaggtatgca aagcccatat ccaatg 36

<210> 25

<211> 34

<212> DNA

<213> Artificial Synthesis

<400> 25

accaggtctc acgattcagt ccatgtgtgg gaag 34

<210> 26

<211> 59

<212> DNA

<213> Artificial Synthesis

<400> 26

accaggtctc aaggtatggt atggtatgat cataacacgc attctgaaaa tgttatctg 59

<210> 27

<211> 59

<212> DNA

<213> Artificial Synthesis

<400> 27

accaggtctc acgatttaag atggtgtatt atgtcgcctt ttgcaatact ttaatttct 59

<210> 28

<211> 35

<212> DNA

<213> Artificial Synthesis

<400> 28

accaggtctc aaggtatgtg tgaatacagc aaggc 35

<210> 29

<211> 40

<212> DNA

<213> Artificial Synthesis

<400> 29

accaggtctc acgatttaaa acccatgttg actcatgatc 40

<210> 30

<211> 52

<212> DNA

<213> Artificial Synthesis

<400> 30

accaggtctc aaggtatgga aatcaagcca gttgaggtta ttgatggcgt tc 52

<210> 31

<211> 59

<212> DNA

<213> Artificial Synthesis

<400> 31

accaggtctc acgatctatg attcagctaa tttagtattt tccatttctt gagtgcatg 59

<210> 32

<211> 55

<212> DNA

<213> Artificial Synthesis

<400> 32

accaggtctc aaggtatggc taacgtagaa aaaccaaacg attgttcagg ctttc 55

<210> 33

<211> 59

<212> DNA

<213> Artificial Synthesis

<400> 33

accaggtctc acgattcaac tgtttccctt tagatgattt tccaaagtgt ggaaatcac 59

<210> 34

<211> 58

<212> DNA

<213> Artificial Synthesis

<400> 34

accaggtctc aaggtatgtc acaggtttgg cataattcga attcgcaatc aaacgatg 58

<210> 35

<211> 59

<212> DNA

<213> Artificial Synthesis

<400> 35

accaggtctc acgatttatt gaatcttagc ccccttgtct gaagattcag tattcccag 59

<210> 36

<211> 45

<212> DNA

<213> Artificial Synthesis

<400> 36

accaggtctc aaggtatgtc tgctgctcct gtccaagaca aagac 45

<210> 37

<211> 55

<212> DNA

<213> Artificial Synthesis

<400> 37

accaggtctc acgattcaag tagcacttgt ccaattattc caatcccata cattg 55

<210> 38

<211> 57

<212> DNA

<213> Artificial Synthesis

<400> 38

accaggtctc aaggtatgaa gctactgtct tctatcgaac aagcatgcga tatttgc 57

<210> 39

<211> 55

<212> DNA

<213> Artificial Synthesis

<400> 39

accaggtctc acgatttact ctttttttgg gtttggtggg gtatcttcat catcg 55

<210> 40

<211> 42

<212> DNA

<213> Artificial Synthesis

<400> 40

accaggtctc aaggtatgac tacagatcct tctgtcaaat tg 42

<210> 41

<211> 41

<212> DNA

<213> Artificial Synthesis

<400> 41

accaggtctc acgattcaaa tagaagagtt ggatttgtcc t 41

<210> 42

<211> 41

<212> DNA

<213> Artificial Synthesis

<400> 42

accaggtctc aaggtatggg tgtcatcaag aagaaaagat c 41

<210> 43

<211> 48

<212> DNA

<213> Artificial Synthesis

<400> 43

accaggtctc acgatttaaa aaaatacagc atcaagatct ccaaattc 48

<210> 44

<211> 52

<212> DNA

<213> Artificial Synthesis

<400> 44

accaggtctc aaggtatgaa cataccacag cgtcaattta gcaacgaaga gg 52

<210> 45

<211> 46

<212> DNA

<213> Artificial Synthesis

<400> 45

accaggtctc acgatctata cactcgcttc tgtcatgctc gagtcc 46

<210> 46

<211> 41

<212> DNA

<213> Artificial Synthesis

<400> 46

accaggtctc aaggtatgct agtcttcgga cctaatagta g 41

<210> 47

<211> 39

<212> DNA

<213> Artificial Synthesis

<400> 47

accaggtctc acgattcaaa aatcaccgtg ctttttgtg 39

<210> 48

<211> 35

<212> DNA

<213> Artificial Synthesis

<400> 48

accaggtctc aaggtatgtc cggtagaggt aaagg 35

<210> 49

<211> 38

<212> DNA

<213> Artificial Synthesis

<400> 49

accaggtctc acgatttaac caccgaaacc gtataagg 38

<210> 50

<211> 40

<212> DNA

<213> Artificial Synthesis

<400> 50

accaggtctc aaggtatgtc attcgacgac ttacacaaag 40

<210> 51

<211> 40

<212> DNA

<213> Artificial Synthesis

<400> 51

accaggtctc aaggtatgtc attcgacgac ttacacaaag 40

<210> 52

<211> 34

<212> DNA

<213> Artificial Synthesis

<400> 52

accaggtctc aaggtatgac caccactgcc caag 34

<210> 53

<211> 34

<212> DNA

<213> Artificial Synthesis

<400> 53

accaggtctc acgattcaaa cctttgcgcc ggtg 34

<210> 54

<211> 40

<212> DNA

<213> Artificial Synthesis

<400> 54

accaggtctc aaggtatggc tggtgaaact tttgaatttc 40

<210> 55

<211> 40

<212> DNA

<213> Artificial Synthesis

<400> 55

accaggtctc acgatttaat caacttcttc catctcggtg 40

<210> 56

<211> 42

<212> DNA

<213> Artificial Synthesis

<400> 56

accaggtctc aaggtatgtc ccgtgaccta caaaaccatt tg 42

<210> 57

<211> 42

<212> DNA

<213> Artificial Synthesis

<400> 57

accaggtctc acgatttaac tgtcatcagc atatgggcca tc 42

<210> 58

<211> 42

<212> DNA

<213> Artificial Synthesis

<400> 58

accaggtctc aaggtatgaa gttccaagtt gttttatctg cc 42

<210> 59

<211> 38

<212> DNA

<213> Artificial Synthesis

<400> 59

accaggtctc acgattcatg ggaaaatgct ttccagag 38

<210> 60

<211> 45

<212> DNA

<213> Artificial Synthesis

<400> 60

accaggtctc aaggtatgtc tgctgttaac gttgcacctg aattg 45

<210> 61

<211> 49

<212> DNA

<213> Artificial Synthesis

<400> 61

accaggtctc acgatttaac caatcaactc accaaacaaa aatggggtg 49

<210> 62

<211> 34

<212> DNA

<213> Artificial Synthesis

<400> 62

accaggtctc aaggtatgca attctctacc gtcg 34

<210> 63

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 63

accaggtctc acgatttaca acaataaagc ggcagc 36

<210> 64

<211> 41

<212> DNA

<213> Artificial Synthesis

<400> 64

accaggtctc aaggtatgca attacattca cttatcgctt c 41

<210> 65

<211> 43

<212> DNA

<213> Artificial Synthesis

<400> 65

accaggtctc acgatttaca ttatagacat gatgattgcc gtc 43

<210> 66

<211> 50

<212> DNA

<213> Artificial Synthesis

<400> 66

accaggtctc aaggtatgtt taagtctgtt gtttattcgg ttctagccgc 50

<210> 67

<211> 56

<212> DNA

<213> Artificial Synthesis

<400> 67

accaggtctc acgatttatt gttttaatag ggtatcgttg tagtgagtag tattcc 56

<210> 68

<211> 35

<212> DNA

<213> Artificial Synthesis

<400> 68

accaggtctc aaggtatggg ctcaaaagta gcagg 35

<210> 69

<211> 49

<212> DNA

<213> Artificial Synthesis

<400> 69

accaggtctc acgattcagc ttgtatctga gaattttctt ttcttattc 49

Claims (10)

1. The saccharomyces cerevisiae strain is named as CE10, and the preservation number of the saccharomyces cerevisiae strain is CGMCC NO.24860.

2. A method for constructing a fermentation strain, comprising integrating a gene of interest into the genome of the s.cerevisiae strain of claim 1; the target gene is selected from MSN2, TIPI, YRB1 or SPII;

wherein the sequence of the MSN2 is shown as SEQ ID NO.1, the sequence of the TIPI is shown as SEQ ID NO.2, the sequence of the YRB1 is shown as SEQ ID NO.3, and the sequence of the SPII is shown as SEQ ID NO. 4.

3. A construction method of a fermentation strain is characterized by comprising the steps of firstly deleting a CAT8 gene in the genome of the saccharomyces cerevisiae strain of claim 1, and then integrating a target gene into the genome of the saccharomyces cerevisiae strain after the CAT8 gene is deleted by taking 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 sequence of the MSN2 is shown as SEQ ID NO.1, the sequence of the TIPI is shown as SEQ ID NO.2, the sequence of the YRB1 is shown as SEQ ID NO.3, and the sequence of the SPII is shown as SEQ ID NO. 4.

4. The method for constructing a fermentation strain according to claim 3, wherein the step of integrating the target gene into the genome of the Saccharomyces cerevisiae strain after the CAT8 gene is deleted comprises: and integrating a linear fragment containing the target gene into the genome of the saccharomyces cerevisiae strain after the CAT8 gene is deleted, and performing overexpression on the target gene.

5. A fermentation strain produced by the method for producing a fermentation strain according to any one of claims 2 to 4.

6. A method for producing bioethanol comprising fermentatively culturing the strain of claim 1 or 5 in a culture system comprising xylose.

7. The method for producing bioethanol according to claim 6, wherein said xylose is present in said culture system at a concentration of 30 to 50g/L.

8. The method for producing bioethanol according to claim 6, wherein said culture system further comprises glucose.

9. The method for producing bioethanol according to claim 8, wherein a concentration of said glucose in said culture system is 70-100g/L.

10. The method for producing bioethanol according to claim 6, wherein the temperature of said fermentation culture is between 30 and 35 ℃.

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