CN113717873B - A kind of multi-tolerant Saccharomyces cerevisiae strain and its construction method and application - Google Patents

Abstract

The invention discloses a multi-tolerant Saccharomyces cerevisiae strain and its construction method and application. The Saccharomyces cerevisiae strain is preserved in the General Microbiology Center of the China Microbiological Strain Management Committee, the preservation name is SEB17, and its preservation number is: CGMCC NO. 22587, the construction method is as follows: take the strain SEB14 as the starting strain, replace the promoter of the functional gene ENA5 of the strain SEB14 with the promoter of TEF1, and construct the strain SEB17. The bacterial strain SEB17 of the present invention has multiple tolerances and has the advantages of being tolerant to multiple environmental stresses such as high ethanol, high temperature, high sugar, and high salt, and can be applied to material fermentation under various environmental stresses.

Description

A kind of multi-tolerant Saccharomyces cerevisiae strain and its construction method and application

technical field

The invention relates to the technical field of bioengineering, in particular to a multi-tolerant Saccharomyces cerevisiae strain and its construction method and application.

Background technique

Facing the increasingly scarce energy and the increasingly serious environmental pollution, it is urgent to develop green and renewable new energy sources. The resource utilization of organic wastes such as straw and waste molasses with large reserves and production volume to produce clean energy fuel ethanol will alleviate the energy and environmental crises at the same time. If ultra-high concentration (VHG) fermentation and high-temperature fermentation can be realized in the industrial production of fuel ethanol, production costs can be significantly reduced, waste water discharge can be reduced, and environmental pollution load can be reduced. Therefore, breeding multi-tolerant industrial Saccharomyces cerevisiae strains that can tolerate multiple environmental stresses such as high ethanol, high temperature, high sugar, and high salt has important economic and environmental significance. At present, the research on the stress resistance of Saccharomyces cerevisiae is mainly focused on the improvement of single tolerance. The main methods of breeding are mutagenesis, domestication, genome rearrangement and genetic engineering, etc. How to improve the multiple tolerance of Saccharomyces cerevisiae at the same time research is very limited. However, compared with single-tolerant yeast, multi-tolerant Saccharomyces cerevisiae can be applied to the fermentation of a variety of practical materials, so it has wider industrial application value.

Contents of the invention

The object of the present invention is to provide a kind of multi-tolerant Saccharomyces cerevisiae strain, this bacterial strain has multi-tolerance, has the advantage of being tolerant to various environmental stresses such as high ethanol, high temperature, high sugar, high salt simultaneously, can be applicable to many Fermentation of materials under various environmental stresses.

In addition, the present invention also provides a construction method and application of a multi-tolerant Saccharomyces cerevisiae strain.

The present invention realizes through following technical scheme:

A multi-tolerant Saccharomyces cerevisiae strain, the Saccharomyces cerevisiae strain is preserved in the General Microbiology Center (CGMCC) of China Microbiological Culture Management Committee, the preservation name is SEB17, and its preservation number is: CGMCC NO.22587, and the classification is named Saccharomyces cerevisiae , The preservation date is May 24, 2021, and the preservation address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, postcode: 100101.

The bacterial strain SEB17 of the present invention has multiple tolerances and has the advantages of being tolerant to multiple environmental stresses such as high ethanol, high temperature, high sugar, and high salt, and can be applied to material fermentation under various environmental stresses.

A method for constructing a multi-tolerant Saccharomyces cerevisiae strain, using the industrial strain SEB14 with good tolerance as the starting strain, and replacing the promoter of the functional gene ENA5 with the promoter of TEF1, which has a high expression level and is stable under various stresses Son, get high expression gene ENA5 strain SEB17.

Studies have shown that knocking out or overexpressing some transcription factors or functional genes through genetic engineering can effectively improve the tolerance of acetic acid and furfural in Saccharomyces cerevisiae. Therefore, directional transformation of Saccharomyces cerevisiae is very important for improving its tolerance . Based on the research of transcriptomics in the early stage of the present invention, it was found that under various stress conditions such as high ethanol, high temperature, high sugar and high salt, the gene ENA5 (encoding P-type ATPase) in the industrial Saccharomyces cerevisiae strain SEB14 was significantly up-regulated, and the gene most closely related to each gene. At present, only studies have shown that ENA5 is closely related to the salt tolerance of fungi. In the present invention, it is first found that ENA5 is related to the multiple stress tolerance of Saccharomyces cerevisiae.

specifically:

Starting strain:

The starting strain SEB14 was deposited in the General Microorganism Center of China Committee for the Collection of Microbial Cultures with the preservation number CGMCCNo.19587.

Medium:

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

Table 1 Medium Composition

Plasmids, strains and primers:

Taking the industrial strain SEB14 with good tolerance (strain preservation number: CGMCCNo.19587) as the starting strain, through the method of promoter replacement, the gene ENA5 was overexpressed to obtain the strain SEB17, wherein the plasmids and strains used are shown in Table 2; The primers and fragments used for transformation are shown in Table 3; the required homologous wall sequence and the 20bp target sequence upstream of the PAM site (NGG) used to construct the gRNA plasmid are shown in Table 4.

Plasmids and strain information used in the process of constructing bacterial strain SEB17 in table 2

Table 3 Primers and fragments required for strain transformation

Note: TG: gRNA plus homology arm primer, RF: repair fragment, Vp: verification primer; F: upstream primer; R: downstream primer

The sequence number of TEF1 F is SEQ ID No.1, the sequence number of TEF1 R is SEQ ID No.2, the sequence number of ENA5 TG F is SEQ ID No.3, the sequence number of ENA5 TG R is SEQ ID No.4, The serial number of ENA5 RF F is SEQ ID No.5, the serial number of ENA5 RF R is SEQ ID No.6, the serial number of ENA5 V _P F is SEQ ID No.7, and the serial number of ENA5 V _P R is SEQ ID No. .8, the sequence number of Cas9-dg-F is SEQ ID No.9, the sequence number of Cas9-dg-R is SEQ ID No.10, the sequence number of 6006-F is SEQ ID No.11, and the sequence number of 6005-R It is SEQ ID No.12.

Table 4 constructs the required homology arm sequence of gRNA and the 20bp target sequence upstream of the PAM site (NGG)

Note: N20: 20bp target sequence upstream of the PAM site (NGG) contained in the gRNA; underline: PAM site (NGG)

The sequence number of tgR F is SEQ ID No.13, the sequence number of tgR R is SEQ ID No.14, and the sequence number of ENA5 is SEQ ID No.15.

Construction of strain SEB17:

1), amplification of TEF1 promoter (repair fragment)

Using the genomic DNA of strain SEB14 as a template, the TEF1 promoter (repair fragment) was amplified by PCR (Table 5). The PCR reaction system and reaction conditions are shown in Table 6. RFF/RFR is a primer for TEF1 containing a homology arm, and its sequence is shown in Table 3. The PCR product was electrophoresed on 1.5% agarose gel (100V, 32min), stained in EB staining solution for 40min, and the target band was observed under ultraviolet light. The resulting PCR products were stored in a -20°C refrigerator for future use.

Table 5 TEF1 promoter gene sequence list

Table 6PCR amplification TEF1 promoter (repair fragment)

2), construction of double-stranded gRNA fragments:

Query the base sequence of the promoter region of the target gene on the Saccharomyces Genome Database website. Then input the sequence into Yeastriction to search for the 20bp target sequence upstream of the PAM site (NGG) on the target fragment. Synthesize gRNA fragments containing upstream and downstream 50bp homology arms and 20bp target sequences (total 120bp) (see Table 3 for fragment sequences. After synthesis, dilute to 10μM with sterile water, and then dilute the two complementary strand solutions by 1: 1 mix (volume ratio), and heat shock at 95°C for 5 minutes in a water bath to obtain double-stranded gRNA fragments.

3), amplified gRNA linear backbone

Using the pMEL13 plasmid as a template to amplify the linear backbone of gRNA, the PCR reaction system and reaction conditions are shown in Table 7. The PCR product was electrophoresed on 1.5% agarose gel (100V, 32min), stained in EB staining solution for 40min, and observed under ultraviolet light to see if there was a target band. The resulting PCR products were stored in a -20°C refrigerator for future use.

Table 7PCR amplification gRNA linear backbone

4), Purified repair fragment (TEF1 promoter) and gRNA linear backbone

PCRPurification Kit纯化试剂盒进行纯化。具体步骤如下：Since the PCR amplification product contains template, DNA polymerase and buffer, it is necessary to use

PCR Purification Kit purification kit for purification. Specific steps are as follows:

(1) Take 50-70 μL of PCR product, add 5 times the volume of solution BB to mix, add to the spin column, centrifuge at 10000×g for 1 min, and discard the effluent;

(2) Add 650 μL of solution WB, centrifuge at 10,000×g for 1 min, and discard the effluent;

(3) Centrifuge at 10000×g for 2 minutes to completely remove residual WB;

(4) Place the spin column in a 1.5mL centrifuge tube, add 30-50μL sterilized water to the center of the column, let stand at room temperature for 1-2min, and centrifuge at 10000×g for 1min to elute the DNA. The resulting DNA was stored at -20°C.

5), gRNA linear backbone template digestion

The enzymatic digestion system and reaction conditions are shown in Table 8. The amount of Quick cut Dpn1 added in the reaction system is determined according to the amount of pMEL13 template added when amplifying the gRNA linear backbone. The gRNA linear backbone is obtained after purification.

Table 8 gRNA linear backbone template digestion system

6), Gibson connects the gRNA fragment and the gRNA linear backbone

The obtained gRNA fragments and gRNA linear backbone were subjected to Gibson ligation by the method described in the Gibson Assembly kit, and the reaction system is shown in Table 9. gRNA (120bp) and pMEL13-bachbone were added to the ligation system at a mass ratio of 5:1. The reaction system solution was placed on a PCR instrument, and ligated at 50°C for 15 minutes. All the reaction solution was transformed into Escherichia coli. Then, spread the bacterial solution on the LB-Amp antibiotic plate. After the strains grew out, they were streaked on LB-Kana antibiotic plates and cultured at 37°C for 24h. The clones were transferred to a test tube containing 5mL LB+Kana liquid medium and cultured for 12-16h (160rpm, 37°C). The bacteria were collected, and the gRNA plasmid was extracted from Escherichia coli using the SanPrep column plasmid DNA mini-extraction kit. Using 1.5% agarose gel, the gRNA plasmid was electrophoresed (100V, 32min), stained in EB for 40min, and observed under ultraviolet light to see if there was a target band. The resulting gRNA plasmid was sequenced and confirmed to obtain a gRNA plasmid with the correct sequence.

Table 9 The reaction system of Gibson connection

7), Cas9 plasmid

The Escherichia coli cells containing the Cas9 plasmid (Case9-NAT) were taken out from the -80°C refrigerator, streaked on the LB+NAT solid plate, and cultured in a constant temperature incubator at 37°C for 1 day. Pick the bacteria with a toothpick and culture them in 5mL LB+NAT liquid medium for 12-16h (160rpm, 37°C). The bacteria were collected, and the Cas9 plasmid was extracted from Escherichia coli using the SanPrep column plasmid DNA mini-extraction kit. The Cas9 plasmid was electrophoresed on 1.5% agarose gel (100V, 32min), stained in EB staining solution for 40min, and the bands were observed under ultraviolet light.

8), Cas9 plasmid into the target Saccharomyces cerevisiae strain

(1) Activate the target bacterial strain on a 2% YPD solid plate for 24 hours, inoculate an appropriate amount of bacteria into 5 mL of 2% YPD liquid medium and cultivate it for 16 hours (160 rpm, 30° C.);

(2) Take 2-3mL bacterial liquid into 300mL 2% YPD liquid medium, culture at 29℃, 180rpm for 2-3h. During this period, samples were taken every 1 hour, centrifuged at 8,000×g for 2 minutes, and the culture medium was removed. After the bacteria were dispersed with 0.05mmol/L EDTA-2Na solution, the absorbance at 600nm was measured;

(3) When the OD ₆₀₀ reaches 0.2-0.3, centrifuge at 8,000×g for 2 minutes to collect the bacteria, wash the bacteria 2-3 times with sterilized water, and discard the supernatant by centrifugation. Then disperse the bacteria with 0.6mL sterilized water, and put it on ice for later use;

(4) Take the prepared salmon sperm DNA (derived from salmon testes), heat it in a boiling water bath for 5 minutes, put it on ice immediately, and set aside;

(5) Take a 1.5 mL centrifuge tube and add 60% PEG4000 (110 μL), 4M lithium acetate solution (5 μL) and salmon sperm DNA (12 μL) into it respectively. Add 100ng Cas9 plasmid to the experimental group, add the same amount of sterile water to the control group, shake and mix;

(6) Add 50 μL of the host cells prepared in step (3) to the above centrifuge tube, shake and mix;

(7) Heat shock in a metal bath at 42°C for 40-60 minutes, during which the centrifuge tubes were taken out every 20 minutes and turned upside down;

(8) Centrifuge at 8,000×g for 2 minutes, and discard the transformation solution. Wash the cells 2-3 times with sterilized water, then add 1mL 2% YPD liquid medium to the centrifuge tube, and incubate on a shaker at 30°C and 160rpm for 4h;

(9) Centrifuge at 8,000×g for 2 minutes, discard the culture medium, and wash 2-3 times with sterile water. Add 1 mL of sterilized water to disperse the bacteria, take 100 μL of the bacterial suspension, spread it on a 2% YPD plate containing 0.005% NAT, and place it in a constant temperature incubator at 30°C for 1 to 2 days.

9), Cas9 plasmid transformation colony PCR verification:

Pick a single transformant on the 2% YPD+NAT plate, streak it on the 2% YPD+NAT solid plate, culture at 30°C for 24 hours, and perform colony PCR verification. Specific steps are as follows:

(1) From the 2% YPD+NAT plate, pick an appropriate amount of bacteria in a 1.5mL centrifuge tube containing 95 μL of 1% SDS and 5 μL of 4mol/L lithium acetate solution, and vortex;

(2) Heat shock at 75°C for 10 minutes;

(3) Add 300 μL of absolute ethanol to the centrifuge tube, and vortex;

(4) Centrifuge at room temperature at 13,000 rpm for 3 minutes, discard the supernatant, and dry at 37°C for 10 minutes;

(5) Add 100 μL of sterilized water, vortex, and centrifuge at room temperature at 13,000 rpm for 1 min;

(6) Take more than 1 μL of the supernatant as a template for PCR verification, and the PCR reaction system and reaction conditions are shown in Table 10;

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

Table 10 Cas9 plasmid verification PCR system

10), yeast transformation

The specific method of yeast transformation is the same as 8), the Cas9 plasmid is transferred into the target Saccharomyces cerevisiae strain, but the step (5) is changed to: take several 1.5mL centrifuge tubes, add 60% PEG4000 (240μL) and 4M lithium acetate to them respectively (9μL) and salmon sperm DNA (25μL). Add gRNA plasmid (300-600ng) and repair fragment (600-1400ng) to the experimental group, add the same amount of sterilized water to the control group, shake and mix.

11), target strain transformation colony PCR verification

Randomly pick transformants from the 2% YPD+NAT+G418 plate, streak on the 2% YPD+NAT+G418 solid plate, culture at 30°C for 24 hours, refer to the method in the PCR verification of the Cas9 plasmid transformed colony for colony PCR verification. Using the verified primers (Table 3) of the target gene, amplify the target fragment. Among them, the PCR reaction system and reaction conditions are shown in Table 10, and the annealing temperature and fragment extension time are determined by specific verification primers.

12), plasmid removal

After colony PCR verification, the correct transformants were selected, and all Cas9 plasmids and gRNA plasmids were removed.

Specific steps are as follows:

(1) Pick the correct transformant and streak it on a 2% YPD solid plate, and culture it at 30°C for 24h;

(2) Inoculate the bacteria into 5 mL of 2% YPD liquid medium, and culture at 30°C and 160 rpm for 16 hours;

(3) Take 1mL of bacterial liquid, centrifuge at 8000×g for 2min, and discard the supernatant;

(4) Gradiently dilute 10 ^{to 5} times with sterilized water, take 100 μL of the bacterial solution and spread it on a 2% YPD solid plate, and incubate in a constant temperature incubator at 30°C for 1 day;

(5) Pick a single colony and spot it on a plate with 2% YPD, 2% YPD+NAT and 2% YPD+G418 in turn, and culture it in a constant temperature incubator at 30°C for 1-2 days. If the colony can only grow on 2% YPD, it means that the plasmid in the colony has been removed.

The bacterial agent prepared by strain SEB17.

The application of a multi-tolerant brewer's yeast strain in the preparation of bacterial agents.

Application of a multi-tolerant Saccharomyces cerevisiae strain in material fermentation under stress conditions.

Further, the stress condition includes at least one of high ethanol, high temperature, high sugar and high salt.

High ethanol in this aspect is: 15% YPD+7%-10% initial ethanol; high temperature: 42-44°C; high sugar: 250-300g/L; high salt: 1-1.5mol/L.

Application of a multi-tolerant Saccharomyces cerevisiae strain in the fermentation of organic materials.

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

1. The strain SEB17 of the present invention has multiple tolerances, and has the advantages of being tolerant to various environmental stresses such as high ethanol, high temperature, high sugar, and high salt, and can be applied to material fermentation under various environmental stresses. Compared with bacterial strain SEB14 , all aspects of tolerance have been improved.

2. The present invention found for the first time that the highly expressed gene ENA5 can significantly improve the multiple tolerances such as ethanol resistance, high temperature resistance and osmosis resistance of the industrial Saccharomyces cerevisiae strain SEB14. Fermentation in typical real materials such as straw, waste molasses and cassava all have higher ethanol yields, showing good potential for industrial application.

Description of drawings

The drawings described here are used to provide a further understanding of the embodiments of the present invention, constitute a part of the application, and do not limit the embodiments of the present invention. In the attached picture:

Fig. 1 is the comparison chart of bacterial strain SEB17 and SEB14 ethanol glucose co-fermentation result;

Fig. 2 is a comparison chart of the high-temperature fermentation results of bacterial strains SEB17 and SEB14;

Fig. 3 is a comparison chart of the high-temperature ethanol co-fermentation results of bacterial strains SEB17 and SEB14;

Fig. 4 is the comparison chart of bacterial strain SEB17 and SEB14 VHG fermentation result;

Fig. 5 is a comparison chart of high-salt fermentation results of bacterial strains SEB17 and SEB14;

Figure 6 is a comparison of the results of simultaneous saccharification and fermentation of strains SEB17 and SEB14 using pretreated straw;

Figure 7 is a comparison chart of the VHG fermentation results of strains SEB17 and SEB14 utilizing waste molasses;

Fig. 8 is a comparison chart of simultaneous saccharification and fermentation results of strains SEB17 and SEB14 using cassava materials.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples and accompanying drawings. As a limitation of the present invention.

Example 1:

Ethanol glucose co-fermentation:

Using the industrial Saccharomyces cerevisiae strain SEB14 as the host and highly expressing the gene ENA5, an excellent multi-tolerant strain SEB17 was obtained. After co-fermentation with 9% (v/v) initial ethanol and 141.95g/L glucose for 96h, it produced 32.39±1.02g/L ethanol, which was 13.09% (28.64±1.66g/L) higher than that of strain SEB14. Meanwhile, after 24 hours of fermentation, the fermentation rate of SEB17 was significantly higher than that of SEB14. The results indicated that the highly expressed gene ENA5 could significantly improve the ethanol tolerance of SEB14, as shown in Table 11 and Figure 1 .

Table 11 Comparison of strain fermentation results for 96h under the mixed conditions of 9% (v/v) ethanol and 141g/L glucose

Note: The same letter in the same column means no significant difference, different letters mean significant difference, P<0.05(t-test). 9% (v/v) ethanol: the initial medium contains 64.17g/L. SEB17: strain SEB14 highly expressed gene ENA5.

Example 2:

High temperature fermentation:

In this example, the high temperature tolerance of strain SEB17 was explored. Under the fermentation conditions of 44°C and 100.65g/L glucose, the ethanol concentration of strain SEB17 was significantly (P<0.05) higher than that of SEB14 (Figure 2). After 72 hours of fermentation, the ethanol concentration and glucose concentration of SEB17 were 43.52±1.17g/L and 7.21±2.03g/L, respectively. Compared with SEB14, it increased by 14.17% and decreased by 59.74% (Table 12). This result indicated that high expression of ENA5 could significantly improve the high temperature tolerance of strain SEB14.

Table 12 Comparison of results of strains fermented for 72 hours at 44°C

Note: The same letter in the same column means no significant difference, different letters mean significant difference, P<0.05(t-test). SEB17:

The strain SEB14 highly expressed the gene ENA5.

Example 3:

High temperature ethanol co-fermentation:

In the late stage of SSF fermentation, yeast cells must tolerate both high temperature and gradually increasing ethanol concentration. In this example, it was found that SEB17 also exhibited better fermentation performance than the starting strain SEB14 under the co-fermentation conditions of initial addition of 3% ethanol and high temperature of 43°C. At the end of the fermentation, its ethanol production concentration (23.43 ± 0.95g/L) increased by 20.36% compared with the starting strain SEB14 (18.66 ± 1.62g/L), indicating that the strain SEB17 can well tolerate the dual stress of high temperature and ethanol, The highly expressed gene ENA5 is crucial for improving the tolerance of strain SEB14 to the double stress of high temperature and ethanol (Fig. 3, Table 13).

Table 13 Comparison of results of 72h fermentation of strains with initial addition of 3% ethanol and 43°C

Note: The same letter in the same column means no significant difference, different letters mean significant difference, P<0.05(t-test). 43°C + 3% ethanol: the initial medium is 21.28g/L ethanol. SEB17: strain SEB14 highly expressed gene ENA5.

Example 4:

VHG fermentation:

In order to further explore the high-sugar tolerance of the strain SEB17, a fermentation study under high-sugar conditions was carried out in this example. Under 270.91g/L glucose fermentation conditions (initial OD ₆₆₀ was 1.5), both strain SEB14 and strain SEB17 could consume glucose within 72h. When the glucose concentration increased to 280.19g/L, the ethanol concentration of the strain SEB17 was not significantly different from that of the starting strain SEB14 24 hours before fermentation. After 48 hours of fermentation, there was a certain difference. After 96 hours of fermentation, the ethanol concentration of SEB17 increased by 3.50% compared with the starting strain SEB14 (Figure 4). The ethanol yields of the two strains both reached 0.50, indicating that they have a strong ability to metabolize ethanol (Table 14). The above results indicated that the highly expressed gene ENA5 could improve the high glucose tolerance of the strain to a certain extent.

Table 14 Comparison of strain fermentation results for 96h under 280.19g/L glucose condition

Note: The same letter in the same column means no significant difference, different letters mean significant difference, P<0.05(t-test). SEB17: strain SEB14 highly expressed gene ENA5.

Example 5:

High salt fermentation:

In this example, it was found that except under high sugar conditions, in the YPD fermentation medium containing 1.25mol/L (7.31%) NaCl, the strain SEB17 could consume all the glucose at 72h, 24h earlier than SEB14. When the NaCl concentration was increased to 1.5mol/L (8.77%), there was no significant difference in ethanol concentration between SEB17 and SEB14 24 hours before fermentation, and after 48 hours of fermentation, the grape consumption rate and ethanol production of SEB17 were significantly higher than those of SEB14 (Figure 5). After 96 hours of fermentation, the end-point ethanol concentration of SEB17 reached 56.73±1.05g/L, which was 101.67% higher than that of SEB14 (28.13±1.58g/L) (Table 15).

Table 15 Comparison of strains fermented for 96h under 1.5M NaCl condition

Note: The same letter in the same column means no significant difference, different letters mean significant difference, P<0.05(t-test). SEB17: strain SEB14 highly expressed gene ENA5.

Embodiment 6:

Pretreatment Straw Fermentation:

my country can produce billions of tons of waste crop straw every year, and the production of fuel ethanol from straw is an important direction for the development of renewable and clean liquid fuels in my country. As shown in Figure 6, after 8 hours of saccharification of the straw pretreated material, during the high-temperature simultaneous saccharification and fermentation process at 42°C, along with the enzymatic hydrolysis process, the glucose in the fermentation broth was quickly utilized by the yeast 24 hours before fermentation, and after 48 hours of fermentation, The ethanol concentration of strain SEB17 was higher than that of SEB14, and the final ethanol concentration reached 68.40±1.59g/L.

Embodiment 7:

Waste molasses raw material fermentation:

Waste molasses is the main by-product produced in the sugar cane crushing process. It has high sugar content and low cost. It has become one of the main raw materials for producing fuel ethanol in Brazil, India and other countries. Under the condition of 270.91g/L total sugar, 48h before fermentation, the ethanol concentration of strains SEB17 and SEB14 had little difference, reaching 94-96g/kg. After 96 hours of fermentation, the ethanol concentration of strain SEB17 was 98.28±2.47g/L, which was higher than that of strain SEB14 (95.71±3.91g/L) ( FIG. 7 ).

Embodiment 8:

Cassava is known as the "king of starch" because of its strong adaptability, high yield and high starch content, and it is a starchy biomass energy crop with great potential. It can be seen from Figure 8 that under the condition of 35% solid content, the ethanol production rate of the strains SEB17 and SEB14 was relatively fast 24 hours before fermentation, and the ethanol production concentration of the strains tended to be stable after 48 hours of fermentation. (138.43±2.06g/L) was higher than the starting strain SEB14 (134.70±1.87g/L).

In summary, the present invention found for the first time that the highly expressed gene ENA5 can significantly improve the multiple tolerances of industrial Saccharomyces cerevisiae strain SEB14, such as ethanol resistance, high temperature resistance and osmosis resistance. And fermentation in typical real materials such as pretreated straw, waste molasses and cassava all have higher ethanol yields, showing good potential for industrial application.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the scope of the present invention. Protection scope, within the spirit and principles of the present invention, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.

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Claims (6)

1. A multi-tolerant Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) bacterial strain is characterized in that, said Saccharomyces cerevisiae bacterial strain is preserved in China Microbiology Strain Management Committee Common Microorganism Center, and preservation name is SEB17, and its preservation number is: CGMCC NO. 22587. 2. the construction method of a kind of multi-tolerant saccharomyces cerevisiae strain as claimed in claim 1, is characterized in that, take bacterial strain SEB14 as departure bacterial strain, the promoter of the functional gene ENA5 of bacterial strain SEB14 is replaced with the promoter of TEF1, The strain SEB17 was constructed; the starting strain SEB14 was preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms, and the preservation number is CGMCCNo.19587. 3. the inoculant prepared by adopting a kind of multi-tolerant Saccharomyces cerevisiae strain described in claim 1. 4. The application of a kind of multi-tolerant Saccharomyces cerevisiae strain as claimed in claim 1 in the preparation of inoculum. 5. the application of a kind of multi-tolerant saccharomyces cerevisiae bacterial strain as claimed in claim 1 in material fermentation under stress condition; Stress condition is the dual stress of high temperature and ethanol, ethanol stress, high-salt stress, high-temperature stress or high sugar coercion. 6. The application of a kind of multi-tolerant Saccharomyces cerevisiae strain as claimed in claim 1 in the fermentation of organic material.

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