CN113717874B

CN113717874B - High-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain as well as construction method and application thereof

Info

Publication number: CN113717874B
Application number: CN202111137904.5A
Authority: CN
Inventors: 汤岳琴; 王莉; 谢采芸; 苟敏; 孙照勇; 夏子渊
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2023-04-11
Anticipated expiration: 2041-09-27
Also published as: CN113717874A

Abstract

The invention discloses a high temperature resistant and high sugar resistant saccharomyces cerevisiae strain and a construction method and application thereof, wherein the saccharomyces cerevisiae strain SEB19 is preserved in the China general microbiological culture center of the Committee for culture of microorganisms with the preservation name of SEB19, and the preservation number of the saccharomyces cerevisiae strain SEB19 is as follows: CGMCC NO.22589, the construction method comprises the following steps: the strain SEB4 is used as an original strain, and a functional gene ASP3 of the strain SEB4 is knocked out by using a CRISPR/Case9 gene editing technology to obtain an engineering strain SEB19. The strain SEB19 disclosed by the invention can resist high temperature and high sugar, ensures that the enzymolysis and microbial fermentation of cellulose can be synchronously carried out, and can improve the yield of ethanol.

Description

High-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain as well as construction method and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a high-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain and a construction method and application thereof.

Background

In recent years, with the deterioration of global environment and the increasing depletion of fossil energy, more and more countries have been concerned about the development and utilization of renewable clean energy fuel ethanol. The production of fuel ethanol is usually carried out by fermenting Saccharomyces cerevisiae (Saccharomyces cerevisiae) with an optimal growth temperature of 30-32 ℃. However, in industrial production, there is an important problem in the Simultaneous Saccharification and Fermentation (SSF) process, that is, there is a large difference between the optimal enzymolysis temperature (45-50 ℃) and the optimal microbial ethanol fermentation temperature (25-35 ℃), which makes it difficult to perform the enzymolysis and microbial fermentation processes of cellulose synchronously. Meanwhile, the temperature of a fermentation system is increased (often to 35-37 ℃) along with the metabolism of strains, mechanical stirring and the like in industrial production. High temperature can cause the physicochemical properties of various components in the yeast cells to change, thereby influencing the normal life activities of the cells. In order to reduce the use of cooling water and the risk of contamination, the breeding of high-temperature-resistant saccharomyces cerevisiae can ensure the stability of fuel ethanol production on the premise of wider temperature control, and the cooling cost is reduced. Furthermore, in ultra-high concentration (VHG) fermentation processes, it is desirable that the yeast is able to tolerate high sugar stress to increase ethanol yield.

Therefore, the construction of high temperature resistant and high sugar resistant saccharomyces cerevisiae is necessary and has certain economic value.

Disclosure of Invention

The invention aims to provide a high-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain, which can resist high temperature and high sugar, ensure that the enzymolysis and microbial fermentation of cellulose can be synchronously carried out, and improve the yield of ethanol.

In addition, the invention also provides a construction method and application of the high temperature resistant and high sugar resistant saccharomyces cerevisiae strain.

The invention is realized by the following technical scheme:

a high temperature resistant and high sugar resistant Saccharomyces cerevisiae strain is preserved in China General Microbiological Culture Center (CGMCC), the preservation name is SEB19, and the preservation number is as follows: CGMCC NO.22589, classified and named as Saccharomyces cerevisiae, has a preservation date of 2021, 5 months and 24 days, and a preservation address of: the western road No.1 hospital, north cheng chaoyang district, no.3, in beijing, china, the zip code: 100101.

the strain SEB19 disclosed by the invention has multiple tolerance, can resist high temperature and high sugar, ensures that the enzymolysis and microbial fermentation of cellulose can be synchronously carried out, can improve the yield of ethanol, and can be suitable for material fermentation under various environmental stresses.

A construction method of a high-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain takes an industrial saccharomyces cerevisiae strain SEB4 with good tolerance as a starting strain, and functional genes ASP3 are knocked out by using a CRISPR/Case9 gene editing technology to obtain an engineering strain SEB19.

In the early stage of the invention, researches based on transcriptomics find that the gene ASP3 (coding L-asparaginase II) in the industrial saccharomyces cerevisiae strain SEB4 is obviously reduced under various stress conditions of high temperature, high temperature ethanol double stress, high sugar and the like. At present, only studies have shown that ASP3 expression is induced during nitrogen starvation of yeast cells, and the relationship between this gene and the cell phenotype has not been confirmed. The invention discovers for the first time that the gene knockout can obviously improve the tolerance of the yeast such as high temperature, high temperature ethanol, high sugar and the like.

Specifically, the method comprises the following steps:

starting strains:

the starting strain SEB4 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCCNo.11324.

Culture medium:

the media used are shown in Table 1. If the culture medium is a solid medium, 2.0% agar powder is added before sterilization. The sterilization condition is 121 deg.C, 15min. All antibiotics are added after being cooled to 50-60 ℃ after the culture medium is sterilized.

TABLE 1 Medium composition

Plasmid, strain and primers:

using a strain SEB4 (the strain preservation number is CGMCC No. 11324) as an original strain, knocking out ASP3 by a CRISPR/Cas9 method to obtain a strain SEB19, wherein plasmids and strains used are shown in a table 2; primers and fragments used for strain transformation are shown in Table 3; the required homologous wall sequences and 20bp target sequences upstream of the PAM site (NGG) for constructing gRNA plasmids are shown in table 4.

TABLE 2 plasmid and Strain information used in the construction of Strain SEB19

/>

Note: TG gRNA plus homology arm primer, RF: repair fragment, vp: verifying the primer; f: an upstream primer; r: the sequence number of the downstream primer ASP3 TG F is SEQ ID No.1, the sequence number of the ASP3 TG R is SEQ ID No.2, the sequence number of the ASP3 RF F is SEQ ID No.3, the sequence number of the ASP3 RF R is SEQ ID No.4, and the sequence number of the ASP 3V is SEQ ID No.2 _P The sequence number of F is SEQ ID No.5, ASP 3V _P The sequence number of R is SEQ ID No.6, the sequence number of Cas9-dg-F is SEQ ID No.7, the sequence number of Cas9-dg-R is SEQ ID No.8, the sequence number of 6006-F is SEQ ID No.9, and the sequence number of 6005-R is SEQ ID No.10.

TABLE 4 homology arm sequences required for the construction of gRNAs and 20bp target sequences upstream of the PAM site (NGG)

Note: n20: a target sequence of 20bp upstream of a PAM site (NGG) contained in the gRNA; underlining: PAM locus (NGG)

the sequence number of the tgR is SEQ ID No.11, the sequence number of the tgR is SEQ ID No.12, and the sequence number of the ENA5 is SEQ ID No.13.

Construction of Strain SEB19:

1) Synthesis of repair fragments

The repair fragment of gene ASP3 is upstream 60bp and downstream 60bp of target gene coding sequence, the fragment is synthesized by Jinwei Zhi company (see sequence in Table 3), after synthesis, the fragment is diluted to 10 MuM with sterilized water, and then two complementary strand solutions are mixed according to the ratio of 1:1, mixing (volume ratio), and thermally shocking in a water bath kettle at 95 ℃ for 5min to obtain the repair fragment of the knockout strain.

2) Constructing a double-chain gRNA fragment:

the base sequence of the CDS region of the target gene is searched on the website of Saccharomyces Genome Database. Then inputting the sequence into Yestrastrication, and searching a 20bp target sequence at the upstream of a PAM locus (NGG) on a target fragment. gRNA fragments containing upstream and downstream 50bp homology arms and 20bp target sequences (120 bp total) were synthesized (see Table 3 for fragment sequences). After synthesis, diluted to 10 μ M with sterile water, the two complementary strand solutions were mixed at 1:1 (volume ratio), and thermally shocking in a water bath kettle at 95 ℃ for 5min to obtain double-stranded gRNA fragments.

3) Amplification of gRNA Linear backbone

The linear backbone of gRNA was amplified using pMEL13 plasmid as template, and the PCR reaction system and reaction conditions are shown in table 5. The PCR product was electrophoresed (100V, 32min) on 1.5% agarose gel, stained in EB staining solution for 40min, and observed under an ultraviolet lamp for the presence of a target band. The PCR product was stored in a refrigerator at-20 ℃ for further use.

TABLE 5 PCR amplification of gRNA Linear frameworks

4) Purified gRNA linear frameworks

Since the PCR amplification product contains the template, DNA polymerase and buffer, it is necessary to utilize

The PCR Purification Kit was purified. The method comprises the following specific steps:

(1) Taking 50-70 μ L of PCR product, adding 5 times volume of solution BB, mixing, adding into a centrifugal column, centrifuging at 10000 Xg for 1min, and discarding the effluent.

(2) Adding 650 μ L of WB solution, centrifuging at 10000 Xg for 1min, and discarding the effluent.

(3) Centrifugation at 10000 Xg for 2min completely removed residual WB.

(4) Placing the centrifugal column in a 1.5mL centrifuge tube, adding 30-50 μ L of sterilized water to the center of the column, standing at room temperature for 1-2min, centrifuging at 10000 Xg for 1min, and eluting DNA. The resulting DNA was stored at-20 ℃.

5) gRNA linear skeleton template digestion

The digestion system and reaction conditions are shown in Table 6. The Quick cut Dpn1 added in the reaction system is determined according to the amount of pMEL13 template added when the linear framework of the gRNA is amplified. The linear framework of gRNA is obtained after purification.

TABLE 6 gRNA Linear framework template digestion System

6) Gibson-ligated gRNA fragments and gRNA linear backbones

The resulting gRNA fragments and gRNA linear frameworks were Gibson ligated by the method described in Gibson Assembly, with the reaction system shown in table 7.gRNA (120 bp) and pMEL13-bachbone were mixed at a mass ratio of 5:1 is added to the ligation system. The reaction solution was placed on a PCR instrument and ligated for 15min at 50 ℃. All reaction solutions were taken and transformed into E.coli. Then, the bacterial solution was spread on LB-Amp antibiotic plates. After the strain grows out, the strain is streaked on an LB-Kana antibiotic plate and cultured for 24 hours at 37 ℃. The clone is transferred to a test tube containing 5mL LB + Kana liquid culture medium for 12-16h (160rpm, 37 ℃). Collecting thalli, and extracting gRNA plasmid from escherichia coli by using a SanPrep column type plasmid DNA small extraction kit. The gRNA plasmids were electrophoresed (100V, 32min) on a 1.5% agarose gel, stained in EB for 40min, and observed under an ultraviolet lamp for the presence of a target band. Sequencing and confirming the obtained gRNA plasmid to obtain the gRNA plasmid with correct sequence.

TABLE 7 Gibson-Linked reaction System

7) Cas9 plasmid

The E.coli strain containing Cas9 plasmid (Case 9-NAT) was taken out from the-80 ℃ freezer, streaked on LB + NAT solid plate, and cultured in 37 ℃ incubator for 1d. The thalli are picked up by toothpick and cultured in a liquid culture medium containing 5mL LB + NAT for 12-16h (160rpm, 37 ℃). Collecting thalli, and extracting Cas9 plasmid from escherichia coli by using a SanPrep column type plasmid DNA small extraction kit. Cas9 plasmid was electrophoresed on a 1.5% agarose gel (100V, 32min), stained in EB staining solution for 40min, and bands were observed under UV light.

8) Transferring the Cas9 plasmid into a target saccharomyces cerevisiae strain

(1) The target strain was activated on 2-% YPD solid plates for 24 hours, and an appropriate amount of the cells were inoculated into 5mL of 2-% YPD liquid medium and cultured for 16 hours (160rpm, 30 ℃ C.);

(2) 2-3mL of the bacterial solution was taken to 300mL of 2% YPD liquid medium, and cultured at 29 ℃ and 180rpm for 2-3 hours. Sampling every 1h, centrifuging at 8,000 Xg for 2min, removing culture solution, dispersing thallus with 0.05mmol/L EDTA-2Na solution, and measuring absorbance at 600 nm;

(3) When OD is measured ₆₀₀ When the concentration reaches 0.2-0.3, centrifuging at 8,000 Xg for 2min to collect thallus, washing thallus with sterilized water for 2-3 times, centrifuging and discarding supernatant. Then 0.6mL of sterilized water is used for dispersing the thalli, and the thalli is placed on ice for standby;

(4) Taking prepared salmon sperm DNA (from salmon testis), heating in boiling water bath for 5min, and immediately placing on ice for use;

(5) A1.5 mL centrifuge tube was charged with 60% PEG4000 (110. Mu.L), 4M lithium acetate solution (5. Mu.L) and salmon sperm DNA (12. Mu.L), respectively. Adding 100ng Cas9 plasmid into an experimental group, adding the same amount of sterile water into a control group, and uniformly mixing by shaking;

(6) Adding 50 mu L of the host cells prepared in the step (3) into the centrifuge tube, and uniformly mixing the host cells by oscillation;

(7) Performing heat shock on the metal bath at 42 ℃ for 40-60min, taking out the centrifuge tube every 20min, and turning upside down;

(8) Centrifuge at 8,000 Xg for 2min and discard the transformation solution. Washing the cells with sterile water for 2-3 times, adding 1mL of 2% YPD liquid culture medium into the centrifuge tube, and shake culturing at 30 deg.C and 160rpm for 4h;

(9) Centrifuging at 8,000 Xg for 2min, discarding the culture solution, and washing with sterilized water for 2-3 times. 1mL of sterilized water-dispersed cells were added, 100. Mu.L of the cell suspension was applied to a 2-% YPD plate containing 0.005% of NAT, and the plate was incubated at 30 ℃ for 1 to 2 days in a constant-temperature incubator.

9) And PCR verification of the Cas9 plasmid transformed colony:

individual transformants on YPD + NAT plates at 2% were picked, streaked onto YPD + NAT solid plates at 30 ℃ for 24h, and colony PCR verified. The method comprises the following specific steps:

(1) From the 2% YPD + NAT plate, appropriate amount of the cells were picked up in 1.5mL centrifuge tube containing 95. Mu.L of 1% SDS and 5. Mu.L of 4mol/L lithium acetate solution, and vortexed;

(2) Thermally shocking at 75 deg.C for 10min;

(3) Adding 300 mu L of absolute ethyl alcohol into the centrifuge tube, and carrying out vortex oscillation;

(4) Centrifuging at 13000rpm for 3min at room temperature, removing supernatant, and drying at 37 deg.C for 10min;

(5) Adding 100 mu L of sterilized water, performing vortex oscillation, and centrifuging at 13000rpm at room temperature for 1min;

(6) Taking 1 mu L of the supernatant as a template for PCR verification, wherein the PCR reaction system and the reaction conditions are shown in Table 8;

(7) The PCR product was electrophoresed (100V, 32min) on 1.5% agarose gel, stained in EB staining solution for 40min, and observed under ultraviolet light for the presence of a target band.

TABLE 8 Cas9 plasmid validation PCR System

10 Yeast transformation), yeast transformation

The specific method for yeast transformation is the same as 8), and the Cas9 plasmid is transformed into the target saccharomyces cerevisiae strain, except that the step (5) is changed into the following steps: several 1.5mL centrifuge tubes were each charged with 60% PEG4000 (240. Mu.L), 4M lithium acetate (9. Mu.L) and salmon sperm DNA (25. Mu.L). The experimental group is added with gRNA plasmid (300-600 ng) and repair fragment (600-1400 ng), the control group is added with the same amount of sterilized water, and the mixture is shaken and mixed evenly.

11 PCR verification of transformed colonies of the target strain)

Transformants were randomly picked from 2% assay YPD + NAT + G418 plates, streaked onto 2% assay YPD + NAT + G418 solid plates, cultured for 24h at 30 ℃ for colony PCR validation as described in Cas9 plasmid transformed colony PCR validation. The target fragment was amplified using the validation primers for the target gene (Table 3). Wherein, the PCR reaction system and the reaction conditions are shown in Table 10, and the annealing temperature and the fragment extension time are determined by specific verification primers.

12 Etc.), plasmid removal

And (4) selecting correct transformants through colony PCR verification, and completely removing the Cas9 plasmid and the gRNA plasmid in the transformants. The method comprises the following specific steps:

(1) The correct transformants were selected and streaked onto 2% YPD solid plates and cultured at 30 ℃ for 24h;

(2) Inoculating the cells into a liquid medium containing 5mL2% of YPD, and culturing at 30 deg.C and 160rpm for 16h;

(3) Sucking 1mL of bacterial liquid, centrifuging at 8000 Xg for 2min, and discarding the supernatant;

(4) Gradient dilution with sterile Water 10 ⁵ Doubling, coating 100 μ L of the bacterial solution on a 2% YPD solid plate, and culturing in a 30 ℃ incubator for 1d;

(5) Individual colonies were picked and spotted on plates of 2% YPD, 2% YPD + NAT and 2% YPD + G418 in this order, and cultured in a 30 ℃ incubator for 1-2d. If the colony can only grow on 2% YPD, it is indicated that the plasmid has been removed from the colony.

The bacterial agent is prepared by adopting a strain SEB19.

An application of a high temperature resistant and high sugar resistant saccharomyces cerevisiae strain in preparing a microbial inoculum.

An application of a high-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain in material fermentation under a stress condition.

Further, the stress conditions include at least one of high temperature and high sugar.

The high temperature of the invention is 42-44 ℃, and the high sugar is 250-300g/L.

An application of a high-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain in organic material fermentation.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the strain SEB19 disclosed by the invention can resist high temperature and high sugar, ensures that the enzymolysis and microbial fermentation of cellulose can be synchronously carried out, and can improve the yield of ethanol.

2. According to the invention, the gene ASP3 knockout can be found for the first time, so that the high temperature, high sugar and other tolerance of the industrial saccharomyces cerevisiae strain SEB4 can be obviously improved. Particularly, the knockout of the gene is very important for improving the high-temperature tolerance of the strain, and the invention shows that the strain SEB19 has better application potential in high-temperature synchronous saccharification and fermentation by using actual materials and ethanol production in tropical regions.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a comparative graph of the high-temperature fermentation results of strains SEB19 and SEB 4;

FIG. 2 is a graph comparing the results of high temperature ethanol co-fermentation of strains SEB19 and SEB 4;

FIG. 3 is a graph comparing fermentation results of strains SEB19 and SEB4 VHG;

FIG. 4 is a graph comparing results of simultaneous saccharification and fermentation of strains SEB19 and SEB4 by pretreated straws.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and the accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limiting the present invention.

Example 1:

high-temperature fermentation:

this example explores the high temperature tolerance of strain SEB19, and as shown in fig. 1, the ethanol concentration and glucose consumption rate of strain SEB19 were significantly higher than those of strain SEB4 (fig. 1) throughout fermentation of 100.65g/L glucose at 44 ℃. After fermentation for 72h, the ethanol concentration and the glucose concentration of the SEB19 are respectively 44.37 +/-1.86 g/L and 5.89 +/-2.90 g/L. Compared with SEB4, the yield is improved by 41.35 percent and reduced by 80.04 percent (Table 9). The result shows that the knockout of the gene ASP3 can obviously improve the high-temperature tolerance of the strain SEB 4.

TABLE 9 comparison of results of 72h fermentation of strains at 44 deg.C

Note: the same letter in the same column indicates no significant difference, different letters indicate significant difference, P <0.05 (t-test). SEB19: the gene ASP3 of the strain SEB4 is knocked out.

Example 2:

high-temperature ethanol co-fermentation:

improving the double stress of the yeast on the high-temperature ethanol is very important for the high-efficiency ethanol production in the later stage of SSF fermentation. In the embodiment, the fermentation performance of the strain SEB19 is also superior to that of the original strain SEB4 (figure 2) under the conditions of initial addition of 3% ethanol and high-temperature co-fermentation at 43 ℃. When the fermentation is carried out for 72h, the final ethanol production concentration of SEB19 reaches 25.91 +/-2.24 g/L, and is improved by 99.31% compared with that of the original strain SEB4, and the result shows that the knockout gene ASP3 is important for improving the high-temperature ethanol double stress tolerance of the strain SEB4 (Table 10).

TABLE 10 comparison of results of initial addition of 3% ethanol and fermentation of strains at 43 ℃ for 72h

Note: the same letter in the same column indicates no significant difference, different letters indicate significant difference, P <0.05 (t-test). 43 ℃ plus 3% ethanol the initial medium was 21.28g/L ethanol. SEB19: the gene ASP3 of the strain SEB4 is knocked out.

Example 3:

VHG fermentation:

to further explore the bacteriaThe fermentation performance of strain SEB19 under high-sugar conditions shows that the strain SEB has the fermentation performance under the condition of 270.86g/L glucose (initial OD) ₆₆₀ 1.5), the ethanol concentration of the two strains is not greatly different in the first 24h, the fermentation is carried out for 96h, and the final ethanol concentration of the SEB19 is higher than that of the starting strain SEB4 (figure 3) and reaches 128.19 +/-0.69 g/L. In addition, the ethanol yield of both strains reached 0.50, and the strains had strong ethanol metabolism ability (table 11). The above results show that the ASP3 knockout can improve the high sugar tolerance of the strain to some extent.

TABLE 11 comparison of 96h results for strain fermentation at 270.86g/L glucose

Example 4:

straw pretreatment material pre-saccharification-synchronous saccharification and fermentation:

when the straw pretreatment material is used as a fermentation substrate, as shown in fig. 4, after 8 hours of pre-saccharification, in the synchronous saccharification and fermentation at a high temperature of 42 ℃, the glucose in the fermentation liquid is timely utilized by yeast in the first 24 hours, the ethanol concentration of the strain SEB19 is obviously higher than that of the SEB4, after 48 hours of fermentation, the ethanol concentrations of the two strains tend to be stable, the fermentation time reaches 96 hours, the end point ethanol concentration of the strain SEB19 reaches 65.44 +/-2.94 g/L, and the ethanol concentration is improved by 17.09% (55.89 +/-2.68 g/L) compared with that of the SEB 4.

In conclusion, the gene ASP3 knockout can obviously improve the tolerance of the industrial saccharomyces cerevisiae strain SEB4, such as high temperature, high sugar and the like for the first time. Particularly, the knockout of the gene is very important for improving the high-temperature tolerance of the strain, and the invention shows that the strain SEB19 has better application potential in high-temperature synchronous saccharification and fermentation by using actual materials and ethanol production in tropical regions.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> Sichuan university

<120> high-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain as well as construction method and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 120

<212> DNA

<213> Artificial sequence 1 (Artificial sequence)

<400> 1

tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc gctacggcat 60

ggatcagatt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac 120

<210> 2

<211> 120

<212> DNA

<213> Artificial sequence 2 (Artificial sequence)

<400> 2

gttgataacg gactagcctt attttaactt gctatttcta gctctaaaac aatctgatcc 60

atgccgtagc gatcatttat ctttcactgc ggagaagttt cgaacgccga aacatgcgca 120

<210> 3

<211> 120

<212> DNA

<213> Artificial sequence 3 (Artificial sequence)

<400> 3

agagcaaatg ttggctcgct attcttttgt aagcaatctg gtactcacca acctccaact 60

agcctgatca gtgacttttc atcacactgt gtttttatat agttcttagt agtaaatata 120

<210> 4

<211> 120

<212> DNA

<213> Artificial sequence 4 (Artificial sequence)

<400> 4

tatatttact actaagaact atataaaaac acagtgtgat gaaaagtcac tgatcaggct 60

agttggaggt tggtgagtac cagattgctt acaaaagaat agcgagccaa catttgctct 120

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence 5 (Artificial sequence)

<400> 5

tatcagaccc ttcagcacgt 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence 6 (Artificial sequence)

<400> 6

tgacactgct caagggataa 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence 7 (Artificial sequence)

<400> 7

tcacccttac gttgtttgaa 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence 8 (Artificial sequence)

<400> 8

aggggacgtt atcactcttc 20

<210> 9

<211> 40

<212> DNA

<213> Artificial sequence 9 (Artificial sequence)

<400> 9

gttttagagc tagaaatagc aagttaaaat aaggctagtc 40

<210> 10

<211> 27

<212> DNA

<213> Artificial sequence 10 (Artificial sequence)

<400> 10

gatcatttat ctttcactgc ggagaag 27

<210> 11

<211> 101

<212> DNA

<213> Artificial sequence 11 (Artificial sequence)

<400> 11

tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc ngttttagag 60

ctagaaatag caagttaaaa taaggctagt ccgttatcaa c 101

<210> 12

<211> 101

<212> DNA

<213> Artificial sequence 12 (Artificial sequence)

<400> 12

gttgataacg gactagcctt attttaactt gctatttcta gctctaaaac ngatcattta 60

tctttcactg cggagaagtt tcgaacgccg aaacatgcgc a 101

<210> 13

<211> 23

<212> DNA

<213> Artificial sequence 13 (Artificial sequence)

<400> 13

gctacggcat ggatcagatt agg 23

Claims

1. High-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) The strain is characterized in that the saccharomyces cerevisiae strain is preserved in China General Microbiological Culture Center (CGMCC), the preservation name is SEB19, and the preservation number is as follows: CGMCC NO. 22589.

2. The method for constructing the high temperature and high sugar resistant saccharomyces cerevisiae strain as claimed in claim 1, wherein the strain SEB4 is used as an initial strain, and a CRISPR/Cas9 gene editing technology is used for editing functional genes of the strain SEB4ASP3Knock-out acquisition engineeringStrain SEB19; the starting strain SEB4 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCCNo 11324.

3. The microbial inoculum prepared by the high temperature and high sugar resistant saccharomyces cerevisiae strain of claim 1.

4. The use of a high temperature and high sugar tolerant Saccharomyces cerevisiae strain according to claim 1 in the preparation of a microbial inoculum.

5. Use of a high temperature, high sugar tolerant s.cerevisiae strain according to claim 1 in material fermentation under stress conditions of dual stress of high temperature and ethanol, high temperature stress or high sugar stress.

6. Use of a high temperature, high sugar tolerant saccharomyces cerevisiae strain according to claim 1 in the fermentation of organic materials.