An industrial strain of Saccharomyces cerevisiae and its application in the production of fuel ethanol

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to an industrial strain of Saccharomyces cerevisiae and its application in the preparation of fuel ethanol. Saccharomyces cerevisiae HXL3 was deposited with the China General Microbiology Center under the China Culture Collection Administration on March 15, 2024, under the accession number CGMCC No. 30031. Fermentation of this strain with highly toxic corn straw hydrolysate for 48 hours increased xylose utilization and ethanol production by 69.94% and 11.75%, respectively.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an industrial strain of brewer's yeast and its application in preparing 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 to starch-rich biomasses like cassava, lignocellulose biomass possesses a barrier against degradation. To facilitate enzymatic hydrolysis and fermentation, lignocellulose requires certain pretreatment processes. Typical pretreatment conditions involve high temperature and pressure, which not only produce fermentable glucose and xylose during lignocellulose depolymerization, but also inhibitors that hinder bacterial growth. Inhibitors are primarily classified into three categories: weak acids, furan aldehydes, and phenols. Weak acid inhibitors are primarily acetic acid, followed by formic acid and levulinic acid. Acetic acid originates from the acetyl groups of hemicellulose, while formic acid is produced by 5-hydroxymethylfurfural (5-HMF) and furfural under harsh pretreatment conditions, and levulinic acid is produced by 5-HMF under harsh pretreatment conditions. Representative furan aldehyde inhibitors are 5-HMF and furfural. 5-HMF and furfural are produced during pretreatment from hexoses such as glucose and pentoses such as xylose, respectively. Phenolic inhibitors are the most complex inhibitors, primarily derived from lignin. Due to the complex composition of inhibitors in the hydrolysate, their components cannot be clearly analyzed so far.

[0003] Saccharomyces cerevisiae is recognized worldwide as a promising strain for fuel ethanol fermentation. However, wild-type S. cerevisiae barely utilizes xylose. In recent decades, researchers have generated a number of excellent C6 / C5 co-fermenting S. cerevisiae strains by heterologously expressing xylose isomerase, enhancing the pentose phosphate pathway (PPP), reducing byproduct accumulation, expressing specific xylose transporters, and adaptive evolution. These strains can efficiently ferment glucose and xylose to produce ethanol in inhibitor-free or low-inhibitor media. However, industrial hydrolysates often contain high concentrations of inhibitors, which significantly interfere with S. cerevisiae growth and metabolism. This requires fermentation strains to possess both high xylose utilization and robustness. In recent years, a growing number of studies have revealed an antagonistic relationship between high xylose utilization and robustness in C6 / C5 co-fermenting S. 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 S. cerevisiae). 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 andMolecular Analysis of Antagonism between Xylose Utilization and Acetic AcidTolerance in Glucose / Xylose Cofermentation Saccharomyces cerevisiae Strains. J. Agri. Food Chem. 2025;73:6758–71). Using rational techniques such as transcriptional analysis, a number of genes that can enhance the robustness of Saccharomyces cerevisiae have been discovered. However, after imparting corresponding phenotypes to the strains, most of the improvements were minimal, or enhanced robustness resulted in decreased xylose utilization. No technical methods have been reported to address these issues.

[0004] In the previous work, the highly active xylose isomerase Ru-XI and the enzyme from Meyerozyma guilliermondii The specific xylose transporter MGT05196N360F of Saccharomyces cerevisiae was identified by metabolic engineering and adaptive evolution engineering. A strain of Saccharomyces cerevisiae that efficiently co-utilizes glucose and xylose was constructed. Its growth and metabolism were strongly inhibited under high inhibitory conditions (Hou J, Shen Y, Jiao C, Ge R, Zhang X, Bao X. Characterization and evolution of xylose isomerase screened from the bovinerumen metagenome in Saccharomyces cerevisiae . Journal of Bioscience andBioengineering. 2016;121:160–5; Wang C, Bao X, Li Y, Jiao C, Hou J, Zhang Q, et al. Cloning and characterization of heterologous transportersin Saccharomyces cerevisiaeand identification of important amino acids forxylose 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. BioresourBioprocess. 2016;3:51). Based on this, a mitigating strain, 6M-15, was generated through a combination of mutagenesis and adaptive evolution, demonstrating high xylose utilization and robust antagonism. However, when exposed to the highly toxic hydrolysate produced by a fuel ethanol plant, its growth and metabolism were strongly inhibited by xylose as a carbon source, failing to perform well. Summary of the Invention

[0005] In view of the fact that current brewer's yeast strains for fuel ethanol production have insufficient resistance to natural inhibitors in lignocellulose hydrolysate when using xylose as a carbon source, and the antagonism between the strain's inhibitor resistance and xylose metabolism ability, the present invention provides a C6 / C5 co-fermentation brewer's yeast strain that has both high xylose utilization ability and high robustness, and its application.

[0006] The technical solution of this application is as follows:

[0007] The first purpose of this application is to protect an industrial strain of brewer's yeast, which is named Saccharomyces cerevisiae. Saccharomyces cerevisiae HXL3 was deposited in the General Microbiology Center of China Culture Collection Administration on March 15, 2024, with the deposit number CGMCC No. 30031.

[0008] The second purpose of this application is to protect the use of the above-mentioned industrial strain of Saccharomyces cerevisiae in the preparation of fuel ethanol.

[0009] According to a specific embodiment of the present application, the application includes: fermenting and producing fuel ethanol using highly toxic lignocellulosic biomass hydrolyzate as raw material.

[0010] According to a specific embodiment of the present application, the inhibitor in the highly toxic lignocellulosic biomass hydrolysate includes 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, 4.63-5.35gL -1 Total phenolic compounds.

[0011] According to the specific embodiment of the present application, the acetic acid content can be 4.0 g L -1 , 4.2 g L -1 , 4.4 gL -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 of its components.

[0012] According to the specific embodiment of the present application, the 5-hydroxymethylfurfural content can be 1.90g L -1 , 1.95 gL -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 of its components.

[0013] According to the specific embodiment of the present application, the furfural content can be 0.55g L -1 , 0.60 g L -1 , 0.65g L -1 , 0.70 g L -1 , 0.75g L -1 , 0.80 g L -1 , 0.84 g L -1 or a range of its components.

[0014] According to the specific embodiment of the present application, the total phenolic compound content can be 4.63g L -1、4.70 gL -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 a range of its components.

[0015] According to the specific embodiment of the present application, the fermentation conditions include: initial inoculation OD 600 The value is 3.5; the temperature is 30±5℃.

[0016] The experiment confirmed that strain HXL3 fermented highly toxic corn straw hydrolysate for 48 h, with a xylose utilization rate of 65.75% and an ethanol yield of 49.25 g L -1 , an increase of 69.94% and 11.75% respectively compared with 6M-15.

[0017] Beneficial effects of this application

[0018] The present invention discloses a Saccharomyces cerevisiae strain HXL3 that possesses both high xylose utilization and high robustness. HXL3 demonstrates excellent xylose metabolism and ethanol production in highly toxic corn straw hydrolysate, making it a promising strain for fuel ethanol production. The innovation of this invention lies in addressing the antagonistic relationship between high xylose utilization and high robustness during the breeding of fuel ethanol production strains. This strain's robustness and xylose utilization ability are enhanced through ARTP mutagenesis, expression of H3K23R histone point mutations, and adaptive evolution. After 48 hours of fermentation in highly toxic corn straw hydrolysate, xylose utilization and ethanol yield increased by 69.94% and 11.75%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The adaptive evolution of the strain in 25% pretreatment liquid medium shortened the biomass doubling time;

[0020] Figure 2 Strain A10-1 H3K23R Xylose metabolism performance; A is the base strain A10-1, B is A10-1 H3K23R ;

[0021] Figure 3 Alternating acclimatization with pretreatment solution and xylose medium improved the growth performance of the strain in YPX medium; A was acclimatized for 2 months; B was acclimatized for 6 months;

[0022] Figure 4 Fermentation performance of strain 6M-15 and mutant strain HXL3 in pretreated liquid; A is the starting strain 6M-15, and B is the mutant strain HXL3;

[0023] Figure 5 Fermentation performance of strain 6M-15 and mutant strain HXL3 in highly toxic corn straw hydrolysate; A is the starting strain 6M-15, and B is the mutant strain HXL3;

[0024] Figure 6 Biomass of strain 6M-15 and mutant strain HXL3 in highly toxic corn straw hydrolysate; A is the starting strain 6M-15 and B is the mutant strain HXL3. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to the accompanying drawings and examples. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is merely for the purpose of explaining the present invention and does not limit the present invention in any form. Any simple modifications, equivalent changes, and modifications made to the embodiments based on the technical essence of the present invention fall within the scope of the technical solution of the present invention.

[0026] In the following examples, the materials and reagents used were obtained from commercial sources unless otherwise specified.

[0027] Example 1 Breeding of a C6 / C5 co-fermentation Saccharomyces cerevisiae strain with both high xylose utilization ability and high robustness

[0028] The starting strain is the C6 / C5 co-fermentation Saccharomyces cerevisiae strain 6M-15, which was previously constructed in the applicant's laboratory. This strain has been patented, with patent application number: 202011296559.5 and accession number: CGMCC No. 20436. The specific technical steps are as follows:

[0029] (1) Determine the concentration of the pretreatment liquid acclimation culture medium

[0030] The inhibitor composition of the pretreatment solution is similar to that of the hydrolyzate, with low glucose concentration and high xylose concentration. To ensure that the toxic environment of the acclimation is similar to that of the hydrolyzate, a pretreatment solution with a similar inhibitor concentration as the hydrolyzate was selected as the acclimation medium. Using the pretreatment solution as the acclimation medium is beneficial for improving the robustness of the strain and preserving its ability to utilize xylose.

[0031] Preparation of pretreatment solution and hydrolyzate: After steam explosion pretreatment, corn straw had a dry weight of approximately 37% (g / g). Pretreatment solution of varying concentrations was then prepared. For example, to prepare 100 mL of 20% (m / v) pretreatment solution, 54 g of wet pretreated corn straw was weighed and deionized water was added to 100 mL. After thorough soaking, the supernatant was the 20% pretreatment solution. The pretreated corn straw was hydrolyzed with cellulase at 50°C for 2 days to obtain the hydrolyzate.

[0032] The sample component analysis method is as follows: take 1 mL of sample, high-speed centrifuge (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 high-performance liquid chromatography system Waterse2695 to determine its component content.

[0033] Chromatographic conditions:

[0034] ① Glucose, xylose, acetic acid, and ethanol were analyzed using an HPX-87H ion exclusion chromatography column (Bio-Rad Aminex) at 45°C with 5 mM H2SO4 as the mobile phase and a Waters 2414 RI differential refractive index detector.

[0035] ② Furfural and 5-HMF were separated using a WondaSil C18 column (GL Sciences) at 30°C with acetonitrile: phosphoric acid-sodium dihydrogen phosphate buffer solution (volume ratio of 15:85, pH 2.6) as the mobile phase at a flow rate of 1 mL / min.

[0036] Absorbance was measured at 210 nm using a Waters 2998 PDA UV detector. Total phenolic content in the pretreatment solution was determined using the Folin-Ciocalteu reagent method, using vanillin as a standard. Absorbance was measured at 725 nm. The main sugar components and inhibitor concentrations of the pretreatment solutions and hydrolyzates at different concentrations are shown below (Table 1).

[0037] Table 1 Main sugar components and inhibitor concentrations in pretreatment solution and hydrolyzate

[0038]

[0039] The inhibitors in the prepared pretreatment solutions with mass concentrations of 20%, 25% and 30% were compared with those in the hydrolyzed solution. The results are as follows: Figure 1As shown, the inhibitor concentration in the 25% pretreatment solution was similar to that in the hydrolyzate, so the 25% pretreatment solution was selected as the raw material for the acclimation medium. Saccharomyces cerevisiae thrives in a weakly acidic environment, so the pH of the 25% pretreatment solution was adjusted to 5.5 with ammonia to prepare the acclimation medium. The solid medium for the pretreatment solution was prepared by adding 2% agar powder to the pH-adjusted pretreatment solution.

[0040] (2) ARTP mutagenesis and adaptive evolution of strains (robustness improvement)

[0041] ① Generate a mutant library by inducing 6M-15 in the exponential phase at 10 SLM of gas volume and 120 W of power with induction times of 15, 20, 25, and 30 s, respectively. Incubate the mutant strains in a mixed sugar medium containing glucose and xylose for 30 min to resume growth.

[0042] ② The mutant strain that has recovered growth is acclimated in the acclimation medium of step (1). When the strain is in the exponential growth phase, the bacterial concentration (measured as OD 600 Measure) for transfer and detection of strain OD during culture 600 ( Figure 1 When the doubling time of the strain is no longer shortened, the transfer strategy is changed, that is, when the strain has fully utilized all the glucose and grows on xylose as the carbon source, the transfer is performed, and the OD value of the strain is detected during the culture process. 600 ( Figure 1 When the biomass doubling time of the strain was shortened to about 3 h, the acclimatization was stopped.

[0043] (3) Breeding of highly robust strains

[0044] The bacterial solution obtained in step (2) with shortened biomass doubling time (2) was diluted 10,000-fold and then spread on the pre-treated liquid solid culture medium and cultured in a 30°C incubator. When the colony size was appropriate, larger single colonies were initially screened out. Rescreening was performed using 17.5% pre-treated liquid oxygen-limited fermentation with an inoculum size of 0.028 g DCW / L, cultured in a shaking incubator at 30°C and 200 rpm. The highly robust strain A10-1 was screened out. Simultaneously, a highly robust strain AMMC-1 was screened out using a microbial droplet culture system (MMC).

[0045] The performance of the strain was evaluated using a synergy index that characterizes the balance between high xylose metabolism and high robustness antagonism. RYe / xT (Ratio of ethanol yield from xylose to theoretical value) represents the theoretical value of the actual sugar alcohol conversion rate achieved in a culture medium with xylose as the sole carbon source. A higher value indicates a higher ability of the strain to produce ethanol from xylose. SRS (Survival rate under stress) represents the survival rate of the strain 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 -1 The ratio of the maximum specific growth rate in glucose medium containing vanillin to that in glucose medium is significant. A higher ratio indicates greater robustness. The synergy index (SI) is the product of RYe / xT and SRS. A higher ratio indicates less antagonism between high xylose utilization and robustness.

[0046] As shown in Table 2, the robustness of strains A10-1 and AMMC-1 has been significantly improved. However, their xylose utilization ability has deteriorated. The RYe / xT values ​​of strains 6M-15, A10-1 and AMMC-1 were 0.821, 0.476 and 0.171, respectively, the SRS values ​​were 0.215, 0.473 and 0.503, respectively, and the synergy indexes were 0.177, 0.225 and 0.086, respectively. This shows that strain 6M-15 has a high xylose utilization ability but low robustness. The xylose utilization ability of strain AMMC-1 is severely weakened, and its robustness is high. While strain A10-1 has high robustness, it maintains a certain xylose utilization ability and has the highest SI value. Therefore, strain A10-1 with the highest synergy index was used as a high-robustness strain for the next round of domestication.

[0047] Table 2 Robustness analysis of strains A10-1 and AMMC-1

[0048]

[0049] (4) Breeding of strains with both high xylose utilization ability and high robustness: Expression of the H3K23R mutant gene, which is beneficial for alleviating antagonism, at the histone H3 inoculum of strain A10-1.

[0050] The specific process includes:

[0051] S1 The genome of strain A10-1 was used as a template, and primers RA1-F (sequence shown in SEQ ID No. 1, specifically: 5′-CCTGCGAATCAACCGATACTG-3′) and RA1-R (sequence shown in SEQ ID No. 2, specifically: 5′-CTATGCATTTAGACTGGGGGGAC-3′) were used for PCR amplification to obtain the homology arm RA1 of the fragment.

[0052] S2 was amplified by PCR using primers RA2-F (sequence shown in SEQ ID No. 3, specifically: 5′-gcggatctgccggtctcCGGTGGTTAAACAATCGGTGG-3′) and RA2-R (sequence shown in SEQ ID No. 4, specifically: 5′-GGATTCCATGGGTTTCTGCG-3′) to obtain the homology arm RA2 of the fragment;

[0053] S3 PCR was performed using primers H34-F-RA1 (sequence shown in SEQ ID No. 5, specifically: 5′-GTCCCCCCAGTCTAAATGCATAGCGGTTAGAGCGGATGTGGG-3′) and H3K23R-R (sequence shown in SEQ ID No. 6, specifically: 5′-GCAATTGGCTTCTAAGGCTGCTAGA-3′) to obtain H3K23R-L;

[0054] S4 was amplified by PCR using primers H3K23R-F (sequence shown in SEQ ID No. 7, specifically: 5′-TCTAGCAGCCTTAGAAGCCAATTGC-3′) and H34-R (sequence shown in SEQ ID No. 8, specifically: 5′-acctgcagcgtacgaagcTTAACCACCGAAACCGTACAA GG-3′) to obtain fragment H3K23R-R;

[0055] S4 Using plasmid pUG6-NatR as a template, PCR amplification was performed with primers LnatL-F (sequence shown in SEQ ID No. 9, specifically: 5′-gcttcgtacgctgcaggt-3′) and LnatL-R-RA2 (sequence shown in SEQ ID No. 10, specifically: 5′-gagaccggcagatccgc-3′) to obtain the fragment LoxP-NatR-LoxP. By fusion PCR, the fragments RA1, H3K23R-L, H3K23R-R, LoxP-NatR-LoxP, and RA2 were sequentially ligated to obtain the long fragment RA1-H3K23R-LoxP-NatR-LoxP-RA2.

[0056] S5 Yeast transformation was performed with strain A10-1 (lithium acetate transformation method: colonies were picked from the plate and cultured in 5 mL YPD liquid medium at 30°C; the OD was adjusted the next day). 600 Cultivate to 0.2 for 4-6 h, and wait until OD 600 The cell viability reached 0.6-0.8; 1 mL of bacterial solution was centrifuged to collect the bacteria, and the cells were washed twice with 0.1 M lithium acetate to prepare competent cells; 44 μL of sterile water, 10 μL of fish sperm DNA, and 10 μL of the obtained fragment of S4 were added, mixed, and incubated at 30°C for 30 min; 260 μL of 50% (v / v) PEG and 36 μL of 1 M lithium acetate were added, mixed, and incubated at 30°C for 30 min. Heat shock was performed at 42°C for 15 min; centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and 1 mL of YPD liquid medium was added for incubation for 3 h; the supernatant was discarded by centrifugation, and the cells were washed twice with sterile water; the cells were plated on YPD solid plates supplemented with 100 μg / mL nourseoin (100 μg / mL nourseoin) to integrate the long fragment into the genome to obtain strain A10-1. H3K23R .

[0057] Metabolism in xylose medium showed that the comparison strain A10-1 ( Figure 2 Figure A in the figure), strain A10-1 H3K23R ( Figure 2 The xylose metabolism capacity of the strain (Figure B) did not change, indicating that the H3K23R histone point mutation needs to be combined with adaptive evolution to play an acetylation perturbation role in industrial strains ( Figure 2 In order to maintain the robustness of the strain and enhance its xylose utilization ability, YPX solid medium (solid medium includes: 40 g L -1 Xylose, 10 g L -1 Yeast powder, 20 g L -1 After acclimation for 2 months, the strains were plated on YPX solid medium and colonies with obvious size differences appeared after 48 h of culture ( Figure 3 Select large colonies and acclimate them alternately for 4 months. After culturing for 48 hours, spread them on YPX solid culture medium plates ( Figure 3 Figure B in the middle), the initial screening of larger colonies. Second screening using 17.5% pre-treated liquid medium for oxygen-limited fermentation ( Figure 4 ), the strain performance was tested with an inoculum size of 0.5 g DCW / L. Compared with the starting strain 6M-15 ( Figure 4 Figure A in the figure), mutant strain HXL3 ( Figure 4The mutant strain HXL3 was deposited with the General Microbiology Center of the China Culture Collection Administration (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​March 15, 2024, with the accession number CGMCC No. 30031.

[0058] Example 2 Comparison of the performance of the starting strain 6M-15 and the mutant strain HXL3 in the production of fuel ethanol from corn straw hydrolysate

[0059] (1) Corn straw hydrolyzate culture medium: ingredients are shown in Table 1.

[0060] (2) Activation of strains: Single colonies of the starting strain 6M-15 and the mutant strain HXL3 were picked and inoculated into 5 mL of YPD liquid medium (liquid medium includes: 20 g L -1 Glucose, 10 g L -1 Yeast powder, 20 g L -1 The cells were cultured at 30°C, 200 rpm for 12 h, and then transferred to 30 mL YPD liquid medium for secondary activation for 12 h.

[0061] (3) Fermentation conditions: The initial inoculum size was 0.5 g DCW / L. Ammonia 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.

[0062] The fermentation results showed:

[0063] 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, respectively. -1 ( Figure 5 (A in Figure 2), while those of the mutant strain HXL3 were 65.75% and 49.25 g L -1 ( Figure 5 To monitor growth changes, the supernatant of corn straw hydrolysate was used for fermentation. The results showed that 6M-15 no longer grew when xylose was used as the carbon source ( Figure 6 A in Figure ), 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 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: an initial inoculation OD600 value of 3.5; and a temperature of 30±5°C.

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