Saccharomyces cerevisiae industrial strain and application thereof in preparation of fuel ethanol

Abstract

The invention belongs to the technical field of biology, and particularly relates to a saccharomyces cerevisiae industrial strain and application of the saccharomyces cerevisiae industrial strain to preparation of fuel ethanol. The strain is named as Saccharomyces cerevisiae HXL3, and is preserved in the General Microbiological Center of China Committee for Culture Collection of Microorganisms on March 15, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 30031. When the strain is fermented in high-toxicity corn straw hydrolysate for 48 hours, the utilization rate of xylose and the yield of ethanol are respectively increased by 69.94% and 11.75%.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an industrial strain of Saccharomyces cerevisiae and its application in the preparation of fuel ethanol. Background Art

[0002] The production of fuel ethanol from lignocellulose is of great significance for protecting national energy security and the environment. Compared with high-starch biomass such as cassava, lignocellulose biomass has an anti-degradation barrier. In order to facilitate the enzymatic hydrolysis and fermentation processes, lignocellulose needs to be pretreated to a certain extent. Usually, the pretreatment conditions are high temperature and high pressure, etc. This makes not only fermentable glucose and xylose, etc. generated during the depolymerization of lignocellulose, but also inhibitors that are not conducive to the growth of strains. The inhibitors are mainly divided into three categories: weak acid inhibitors, furfural inhibitors and phenolic inhibitors. The main weak acid inhibitor is acetic acid, followed by formic acid and levulinic acid, etc. Acetic acid comes from the acetyl group of hemicellulose, while formic acid is generated by 5-hydroxymethylfurfural (5-HMF) and furfural under severe pretreatment conditions, and levulinic acid is generated by 5-HMF under severe pretreatment conditions. The representative of furfural inhibitors is 5-HMF and furfural. 5-HMF and furfural are generated from hexoses such as glucose and pentoses such as xylose during pretreatment respectively. Phenolic inhibitors are the most complex inhibitors in composition and mainly come from lignin. Due to the complex composition of inhibitors in the hydrolysis solution, their components cannot be clearly analyzed so far.

[0003] Saccharomyces cerevisiae is globally recognized as the most promising fuel ethanol fermentation strain. However, wild-type Saccharomyces cerevisiae hardly utilizes xylose. In recent decades, through heterologous expression of xylose isomerase in Saccharomyces cerevisiae, strengthening the pentose phosphate pathway (PPP), reducing by-product accumulation, expressing specific xylose transporters and adaptive evolution, etc., a number of excellent C6 / C5 co-fermenting Saccharomyces cerevisiae strains have been obtained, which can efficiently ferment glucose and xylose to produce ethanol in a medium without inhibitors or with low inhibitors. However, most industrial hydrolysis solutions contain relatively high concentrations of inhibitors, which significantly interfere with the growth and metabolism of Saccharomyces cerevisiae. This requires that the fermentation strain simultaneously has high xylose utilization ability and robustness. In recent years, more and more studies have found that there is an antagonistic relationship between the high xylose utilization ability and high robustness of C6 / C5 co-fermenting Saccharomyces cerevisiae (Sun D, Wu L, Lu X, Li C, Xu L, Li H, et al. Engineering transcriptional regulatory networks for improving second-generation fuel ethanol production in Saccharomyces cerevisiae. Synthetic and Systems Biotechnology. 2025;10:207–17; Li C, Yu H, Chen S, Song L, Yuan A, Wei F, et al. Quantification and Molecular Analysis of Antagonism between Xylose Utilization and Acetic Acid Tolerance in Glucose / Xylose Cofermentation Saccharomyces cerevisiae Strains. JAgric Food Chem. 2025;73:6758–71). A number of genes related to improving the robustness of Saccharomyces cerevisiae have been discovered through rational technical methods such as transcriptional analysis. However, after conferring the corresponding phenotypes to the strains, most of the improvements are not obvious, or the robustness is improved while the xylose utilization ability decreases. There is no reported technical method to solve the above problems.

[0004] In previous work, based on the highly active xylose isomerase Ru-XI screened from the bovine rumen metagenome and the specific xylose transporter MGT05196N360F from Meyerozyma guilliermondii , a Saccharomyces cerevisiae strain with efficient co-utilization of glucose / xylose was constructed through metabolic engineering and adaptive evolution engineering, and its growth and metabolism under high inhibitor conditions were strongly inhibited (Hou J, Shen Y, Jiao C, Ge R, Zhang X, Bao X. Characterization and evolution of xylose isomerase screened from the bovine rumen metagenome in Saccharomyces cerevisiae . Journal of Bioscience and Bioengineering. 2016;121:160–5; Wang C, Bao X, Li Y, Jiao C, Hou J, Zhang Q, et al. Cloning and characterization of heterologous transporters in Saccharomyces cerevisiaeand identification of important amino acids for xylose utilization. Metabolic Engineering. 2015;30:79–88; Li H, Shen Y, Wu M, Hou J, Jiao C, Li Z, et al. Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-generation bioethanol production. Bioresour Bioprocess. 2016;3:51). Based on this, a strain 6M-15 with high xylose utilization ability and high robustness against antagonism was obtained by combining mutagenesis and adaptive evolution. However, when faced with the highly toxic hydrolysate prepared in a fuel ethanol factory, its growth and metabolism under xylose carbon source were strongly inhibited and it did not perform well. Summary of the Invention

[0005] Aiming at the insufficient resistance of current fuel ethanol-producing Saccharomyces cerevisiae strains to natural inhibitors in lignocellulosic hydrolysates when using xylose as a carbon source, and the antagonistic phenomenon between strain inhibitor resistance and xylose metabolism ability, the present invention provides a C6 / C5 co-fermenting Saccharomyces cerevisiae strain with both high xylose utilization ability and high robustness and its application.

[0006] The technical solution of this application is as follows: The first object of this application is to protect an industrial Saccharomyces cerevisiae strain named Saccharomyces cerevisiae Saccharomyces cerevisiae HXL3, which was deposited in the "China General Microbiological Culture Collection Center" on March 15, 2024, with the deposit number CGMCC No. 30031.

[0007] The second object of this application is to protect the application of the above-mentioned industrial Saccharomyces cerevisiae strain in the preparation of fuel ethanol.

[0008] According to the specific implementation manner of this application, the above application includes: fermenting and producing fuel ethanol using highly toxic lignocellulosic biomass hydrolysate as a raw material.

[0009] According to the specific implementation manner of this application, the inhibitors in the highly toxic lignocellulosic biomass hydrolysate include 4.00 - 6.2 g L -1 acetic acid, 1.90 - 2.52 g L -1 5-hydroxymethylfurfural, 0.55 - 0.84 g L -1Furfural, 4.63 - 5.35 g / L -1 Total phenolic compounds.

[0010] According to the specific embodiments of the present application, the acetic acid content may be 4.0 g / L -1 、4.2 g / L -1 、4.4 g / L -1 、4.6 g / L -1 、4.8 g / L -1 、5.0 g / L -1 、5.2 g / L -1 、5.4 g / L -1 、5.6 g / L -1 、5.8 g / L -1 、6.0 g / L -1 、6.2 g / L -1 or a range composed thereof.

[0011] According to the specific embodiments of the present application, the 5 - hydroxymethylfurfural content may be 1.90 g / L -1 、1.95 g / L -1 、2.00 g / L -1 、2.05 g / L -1 、2.10 g / L -1 、2.15 g / L -1 、2.20 g / L -1 、2.25 g / L -1 、2.30 g / L -1 、2.35 g / L -1 、2.40 g / L -1 、2.45 g / L -1 、2.50 g / L -1 、2.52 g / L -1 or a range composed thereof.

[0012] According to the specific embodiments of the present application, the furfural content may be 0.55 g / L -1 、0.60 g / L -1 、0.65 g / L -1 、0.70 g / L -1 、0.75 g / L -1 、0.80 g / L -1 、0.84 g / L -1 or a range composed thereof.

[0013] According to the specific embodiments of the present application, the total phenolic compounds content may be 4.63 g / L -1 、4.70 g / L-1 、4.75 g / L -1 、4.80 g / L -1 、4.85 g / L -1 、4.90 g / L -1 、4.95 g / L -1 、5.00 g / L -1 、5.05 g / L -1 、5.10 g / L -1 、5.15 g / L -1 、5.20 g / L -1 、5.25 g / L -1 、5.30 g / L -1 、5.35 g / L -1 or the range composed thereof.

[0014] According to the specific embodiments of the present application, the fermentation conditions include: the initial inoculation OD 600 value is 3.5; the temperature is 30±5°C.

[0015] Experiments confirmed that: when the strain HXL3 fermented the highly toxic corn stover hydrolysate for 48 h, the xylose utilization rate was 65.75%, and the ethanol yield was 49.25 g / L -1 , which were increased by 69.94% and 11.75% respectively compared with 6M-15.

[0016] Advantages of the present application The present invention discloses a Saccharomyces cerevisiae strain HXL3 that simultaneously has high xylose utilization ability and high robustness. It shows excellent xylose metabolism ability to produce ethanol in highly toxic corn stover hydrolysate, and is a promising Saccharomyces cerevisiae dedicated to fuel ethanol production. The innovation of the present invention lies in, aiming at the problem of mutual antagonism between high xylose utilization and high robustness in the breeding process of fuel ethanol production strains, the robustness and xylose utilization ability of the strain are improved by means of ARTP mutagenesis, expressing the histone point mutation H3K23R combined with adaptive evolution. When fermenting in highly toxic corn stover hydrolysate for 48 h, the xylose utilization rate and ethanol yield were increased by 69.94% and 11.75% respectively. Brief description of the drawings

[0017] Figure 1 The strain adapts and evolves in the 25% pretreatment liquid medium to shorten the biomass doubling time; Figure 2 Strain A10-1 H3K23R xylose metabolism performance; where A is the chassis strain A10-1, and B is A10-1 H3K23R ; Figure 3Improving the growth performance of strains in YPX medium by alternating domestication with a pretreatment solution and a xylose medium; where A is domestication for 2 months; B is domestication for 6 months; Figure 4 Fermentation performance of strains 6M-15 and mutant strain HXL3 in the pretreatment solution; where A is the starting strain 6M-15 and B is the mutant strain HXL3; Figure 5 Fermentation performance of strains 6M-15 and mutant strain HXL3 in highly toxic corn stover hydrolysate; where A is the starting strain 6M-15 and B is the mutant strain HXL3; Figure 6 Biomass of strains 6M-15 and mutant strain HXL3 in highly toxic corn stover hydrolysate; where A is the starting strain 6M-15 and B is the mutant strain HXL3. Detailed implementation mode

[0018] The content of the present invention will be described in detail below with reference to specific drawings and examples. The following examples are only the preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not a limitation to the present invention in any form. Any simple modification, equivalent change and modification made to the embodiments according to the technical essence of the present invention all fall within the scope of the technical solution of the present invention.

[0019] In the following examples, the materials, reagents, etc. used are all obtained from commercial channels unless otherwise specified.

[0020] Example 1 Breeding of a C6 / C5 co-fermenting Saccharomyces cerevisiae strain with high xylose utilization ability and high robustness The starting strain is the C6 / C5 co-fermenting Saccharomyces cerevisiae strain 6M-15 constructed by the applicant's laboratory in the early stage. This strain has been patented, with the patent application number: 202011296559.5 and the preservation number: CGMCC No. 20436. The specific technical steps are as follows: (1)Determine the concentration of the domestication medium with the pretreatment solution The inhibitor components of the pretreatment solution are similar to those of the hydrolysate, and it has the characteristics of low glucose concentration and high xylose concentration. In order to make the domesticated toxic environment similar to that of the hydrolysate, the pretreatment solution with inhibitor concentration similar to that in the hydrolysate is selected as the domestication medium. Using the pretreatment solution as the domestication medium is beneficial to improving the robustness of the strain and retaining the xylose utilization ability of the strain.

[0021] Preparation of pretreatment solution and hydrolysis solution: After steam explosion pretreatment of corn stover, the dry weight is about 37% (g / g), and then pretreatment solutions with different concentrations are prepared. Taking the preparation of 100 mL of 20% (m / v) pretreatment solution as an example, 54 g of moist pretreated corn stover is weighed, deionized water is added to make up the volume to 100 mL, and after sufficient infiltration, the supernatant is the 20% pretreatment solution. The pretreated corn stover is hydrolyzed with cellulase at 50 °C for 2 days to obtain the hydrolysis solution.

[0022] The analysis method of sample components is as follows: Take 1 mL of the sample, centrifuge at high speed (13000 rpm, 5 min) to remove impurities or bacteria in the sample, take the supernatant for dilution and filter it with a 0.22 µm microporous filter membrane, and use a Waters e2695 high performance liquid chromatography system to determine its component content.

[0023] Chromatographic conditions: ① Glucose, xylose, acetic acid and ethanol are analyzed using an HPX-87H ion exclusion chromatographic column (Bio-Rad Aminex). This chromatographic column uses 5 mM H2SO4 as the mobile phase at 45 °C and a Waters 2414 RI differential refractive index detector; ② Furfural and 5-HMF are analyzed using a WondaSil C18 chromatographic column (GL Sciences) at 30 °C with acetonitrile: phosphate-sodium dihydrogen phosphate buffer solution (volume ratio 15:85, pH 2.6) as the mobile phase, and the flow rate is maintained at 1 mL / min.

[0024] The absorbance value is detected at 210 nm using a Waters 2998 PDA ultraviolet detector. The total phenol content in the pretreatment solution is detected by the Folin reagent method. Using vanillin as the standard product, it reacts with the Folin-Ciocalteu reagent, and the absorption value is detected at 725 nm. The main sugar components and inhibitor concentrations of the pretreatment solutions and hydrolysis solutions with different concentrations are as shown in Table 1 below.

[0025] Table 1 Main sugar components and inhibitor concentrations of pretreatment solutions and hydrolysis solutions Comparing the inhibitors in the prepared pretreatment solutions and hydrolysis solutions with mass concentrations of 20%, 25% and 30%, the results are as Figure 1 shown. The inhibitor concentration of the 25% pretreatment solution is similar to that of the hydrolysis solution. Therefore, the 25% pretreatment solution is selected as the raw material for the domestication medium. Saccharomyces cerevisiae is suitable to grow in a weakly acidic environment. Therefore, ammonia water is used to adjust the pH of the 25% pretreatment solution to 5.5 as the domestication medium. Preparation method of the pretreatment solution solid medium: Add 2% agar powder to the pretreated solution after adjusting the pH.

[0026] (2)ARTP mutagenesis treatment and domestication adaptive evolution (robustness improvement) of strains ① Set mutagenesis times of 15, 20, 25, and 30 s respectively, and perform mutagenesis on 6M-15 in the exponential phase under the conditions of 10 SLM gas flow and 120 W power to form a mutant library. The mutant strains were incubated in a mixed sugar medium of glucose and xylose for 30 min to resume growth.

[0027] ② The mutant strains that resumed growth were domesticated in the domestication medium in step (1). When the strains were in the exponential phase of growth, the cell concentration was quantified (measured by OD 600 ), and then transferred. During the cultivation process, the OD 600 ( Figure 1 ) of the strains was detected. When the doubling time of the strains no longer shortened, the transfer strategy was changed, that is: when the strains had utilized all the glucose and grew with xylose as the carbon source, they were transferred, and the OD 600 ( Figure 1 ) of the strains was detected during the cultivation process. When the biomass doubling time of the strains was shortened to about 3 h, the domestication was stopped.

[0028] (3) Selection of highly robust strains The bacterial liquid with a shortened biomass doubling time in step (2)② was diluted 10,000 times and spread on the solid medium of the pretreatment solution, and cultured in an incubator at 30 °C. When the colony size was appropriate, larger single colonies were initially screened out. Re-screening was carried out using 17.5% pretreatment solution for oxygen-limited fermentation, with an inoculum amount of 0.028 g DCW / L, cultured at 30 °C on a shaker at 200 rpm. A highly robust strain A10-1 was obtained. At the same time, a highly robust strain AMMC-1 was obtained using a microbial microdroplet culture system (MMC).

[0029] The performance of the strains was evaluated using a synergy index that characterized the balance degree of the antagonistic phenomenon between high xylose metabolism and high robustness. RYe / xT (Ratio of ethanol yield from xylose to theoretical value) represents the theoretical value reached by the actual sugar alcohol conversion rate under the condition of xylose as the sole carbon source medium. The higher it is, the higher the ability of the strain to produce ethanol using xylose. SRS (Survival rate under stress) represents the survival rate in the presence of inhibitors (0.60 g L -1 acetic acid, 0.23 g L -1 formic acid, 0.58 g L -1 levulinic acid, 0.63 g L -1 5-hydroxymethylfurfural, 0.48 g L -1 furfural and 0.76 g L -1The ratio of the maximum specific growth rate of the glucose medium with vanillin to that of the glucose medium, the higher the value, the higher the robustness of the strain. The synergy index SI is the product of RYe / xT and SRS, and the higher the value, the lower the antagonistic relationship between xylose utilization and robustness.

[0030] As shown in Table 2, the robustness of strains A10-1 and AMMC-1 has been significantly improved. However, their xylose utilization abilities have both deteriorated. The RYe / xT values of strains 6M-15, A10-1, and AMMC-1 are 0.821, 0.476, and 0.171 respectively, and the SRS values are 0.215, 0.473, and 0.503 respectively. The synergy indices are 0.177, 0.225, and 0.086 respectively. It shows that strain 6M-15 has a higher xylose utilization ability but lower robustness. Strain AMMC-1 has a severely weakened xylose utilization ability and higher robustness. While strain A10-1 has higher robustness and maintains a certain xylose utilization ability, and its SI value is the highest. Therefore, strain A10-1 with the highest synergy index is used as the highly robust strain for the next round of domestication.

[0031] Table 2 Robustness analysis of strains A10-1 and AMMC-1 (4) Breeding of strains with both high xylose utilization ability and high robustness: Expressing the histone H3 in situ of strain A10-1 is beneficial to alleviating the antagonistic H3K23R mutant gene.

[0032] The specific process includes: S1 Using the genome of strain A10-1 as a template, and performing PCR amplification with primer RA1-F (the sequence is as shown in SEQ ID No.1, specifically: 5′-CCTGCGAATCAACCGATACTG-3′) and primer RA1-R (the sequence is as shown in SEQ ID No.2, specifically: 5′-CTATGCATTTAGACTGGGGGGAC-3′) to obtain the fragment homologous arm RA1.

[0033] S2 Performing PCR amplification with primer RA2-F (the sequence is as shown in SEQ ID No.3, specifically: 5′-gcggatctgccggtctcCGGTGGTTAAACAATCGGTGG-3′) and RA2-R (the sequence is as shown in SEQ ID No.4, specifically: 5′-GGATTCCATGGGTTTCTGCG-3′) to obtain the fragment homologous arm RA2; S3 uses primers H34-F-RA1 (sequence as shown in SEQ ID No.5, specifically: 5′-GTCCCCCCAGTCTAAATGCATAGCGGTTAGAGCGGATGTGGG-3′) and H3K23R-R (sequence as shown in SEQ ID No.6, specifically: 5′-GCAATTGGCTTCTAAGGCTGCTAGA-3′) to perform PCR to obtain H3K23R-L; S4 uses primers H3K23R-F (sequence as shown in SEQ ID No.7, specifically: 5′-TCTAGCAGCCTTAGAAGCCAATTGC-3′) and H34-R (sequence as shown in SEQ ID No.8, specifically: 5′-acctgcagcgtacgaagcTTAACCACCGAAACCGTACAA GG-3′) to perform PCR amplification to obtain the fragment H3K23R-R; S4 uses the plasmid pUG6-NatR as a template and primers LnatL-F (sequence as shown in SEQ ID No.9, specifically: 5′-gcttcgtacgctgcaggt-3′) and primer LnatL-R-RA2 (sequence as shown in SEQ ID No.10, specifically: 5′-gagaccggcagatccgc-3′) to perform PCR amplification to obtain the fragment LoxP-NatR-LoxP. The fragments RA1, H3K23R-L, H3K23R-R, LoxP-NatR-LoxP, and RA2 are sequentially ligated by fusion PCR to obtain the long fragment RA1-H3K23R-LoxP-NatR-LoxP-RA2; S5 performs yeast transformation with strain A10-1 (lithium acetate transformation method: pick colonies from the plate and culture them in 5 mL of YPD liquid medium at 30 °C for expansion; the next day, adjust the OD 600 to 0.2 and culture for 4-6 h until the OD 600 reaches 0.6-0.8; take 1 mL of the bacterial solution, centrifuge to collect the bacteria, wash the cells twice with 0.1 M lithium acetate to prepare competent cells; add 44 μL of sterile water, 10 μL of salmon sperm DNA, and the fragment obtained in S4, mix well and let stand at 30 °C for 30 min; add 260 μL of 50% (v / v) PEG and 36 μL of 1 M lithium acetate, mix well and let stand at 30 °C for 30 min. Heat shock at 42 °C for 15 min; centrifuge at 8000 rpm for 3 min, discard the supernatant, add 1 mL of YPD liquid medium and incubate for 3 h; centrifuge and discard the supernatant, wash the cells twice with sterile water; coat the YPD solid plate supplemented with 100 μg / mL nourseothricin), integrate the long fragment into the genome to obtain strain A10-1H3K23R .

[0034] Metabolic analysis under xylose medium showed that, compared with strain A10-1 ( Figure 2 , Panel A), the xylose metabolic ability of strain A10-1 H3K23R ( Figure 2 , Panel B) did not change, indicating that the H3K23R histone point mutation needs to combine with adaptive evolution to exert the effect of acetylation perturbation in industrial strains ( Figure 2 ). To enhance the xylose utilization ability while maintaining the robustness of the strain, a domestication strategy of alternating cultivation on YPX solid medium (the solid medium includes: 40 g / L -1 xylose, 10 g / L -1 yeast extract, 20 g / L -1 peptone) and 25% pretreated liquid medium was used. After 2 months of domestication, the YPX solid medium plate was coated, and colonies with significantly different sizes appeared after 48 h of cultivation ( Figure 3 , Panel A). Large colonies were selected and domesticated alternately for another 4 months. After 48 h of cultivation, the YPX solid medium plate was coated ( Figure 3 , Panel B), and larger colonies were initially screened out. For the rescreening, oxygen-limited fermentation was carried out using 17.5% pretreated liquid medium ( Figure 4 ), and the strain performance was tested at an inoculum size of 0.5 g DCW / L. Compared with the parental strain 6M-15 ( Figure 4 , Panel A), the growth and xylose metabolic performance of the mutant strain HXL3 ( Figure 4 , Panel B) were improved. The mutant strain HXL3 was deposited in the "China General Microbiological Culture Collection Center (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing)" on March 15, 2024, with the deposit number CGMCC No. 30031.

[0035] Performance comparison between the parental strain 6M-15 and the mutant strain HXL3 in the fermentation of fuel ethanol from corn stover hydrolysate (1) Corn stover hydrolysate medium: The components are shown in Table 1.

[0036] (2) Strain activation: Single colonies of the parental strain 6M-15 and the mutant strain HXL3 were picked and inoculated into 5 mL of YPD liquid medium (the liquid medium includes: 20 g / L -1 glucose, 10 g / L -1 yeast extract, 20 g / L -1 peptone), cultured at 30 °C and 200 rpm for 12 h, and then transferred to 30 mL of YPD liquid medium for secondary activation for 12 h.

[0037] (3) Fermentation conditions: The initial inoculum size was 0.5 g DCW / L. Ammonia water was used to maintain the pH value greater than 4.8 during the fermentation process. Sampling and analysis were performed every 12 h. The detection method was as described in Example 1.

[0038] The fermentation results showed: The mutant strain HXL3 had a better xylose metabolism ability than the original strain 6M-15. After fermentation in highly toxic corn straw hydrolysate for 48 h, the xylose utilization rate and ethanol yield of the original strain 6M-15 were 38.69% and 44.07 g L -1 ( Figure 5 ), while the mutant strain HXL3 was 65.75% and 49.25 g L -1 ( Figure 5 In order to monitor the growth changes, the supernatant of corn stover hydrolysate was used for fermentation. The results showed that 6M-15 no longer grew when xylose was used as the carbon source ( Figure 6 Figure 5A), while HXL3 was able to continue growing ( Figure 6 OD values ​​of 6M-15 and HXL3 600 The highest values ​​were 29.30 and 38.54 respectively.

Claims

1. An industrial strain of Saccharomyces cerevisiae, characterized in that: Saccharomyces cerevisiae Saccharomyces cerevisiae HXL3 was deposited in the "General Microbiology Center of China Microbiological Culture Collection Administration" on March 15, 2024, with the deposit number CGMCC No. 30031.

2. Use of the industrial strain of Saccharomyces cerevisiae according to claim 1 in the preparation of fuel ethanol.

3. The use according to claim 2, characterized in that: The application includes: fermenting and producing fuel ethanol using highly toxic lignocellulose biomass hydrolyzate as raw material.

4. The use according to claim 3, characterized in that The inhibitors in the highly toxic lignocellulosic biomass hydrolysate include 4.00-6.2 g L -1 Acetic acid, 1.90-2.52 g L -1 5-Hydroxymethylfurfural, 0.55-0.84 g L -1 Furfural, 4.63-5.35 g L -1 Total phenolic compounds.

5. The use according to claim 3 or 4, characterized in that: The fermentation conditions included: the initial inoculation OD600 value was 3.5; the temperature was 30±5°C.

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