Saccharomyces cerevisiae capable of efficiently utilizing cellulose hydrolysate as well as construction method and application of saccharomyces cerevisiae

Abstract

The invention discloses saccharomyces cerevisiae capable of efficiently utilizing cellulose hydrolysate as well as a construction method and application thereof. The construction method comprises the following steps: 1) selecting a xylose metabolism saccharomyces cerevisiae haploid chassis strain of a xylose isomerase pathway; 2) performing spore splitting on industrial ethanol-producing yeast to obtain haploid industrial yeast strains; and (3) fusing and screening the strains obtained in the steps (1) and (2) to obtain the saccharomyces cerevisiae capable of efficiently utilizing the cellulose hydrolysate. The saccharomyces cerevisiae. At the shake flask level, 70 g / L of glucose in the acid-treated cellulose hydrolysate can be completely consumed within about 24 h, meanwhile, xylose starts to be consumed gradually, and 20 g / L of xylose in the acid-treated cellulose hydrolysate can be consumed within 72 h. The growth amount OD600 can reach about 15.0 at most, 45 g / L of ethanol is generated under the aerobic condition, rapid growth in industrial cellulose hydrolysate is achieved, glucose and xylose are fully utilized as growth carbon sources, and ethanol is generated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a Saccharomyces cerevisiae for cultivating high-efficiency utilization of cellulose hydrolysate by fusing haploid yeast, and a construction method and application thereof. BACKGROUND

[0002] With the population growth and social development, the over-consumption of traditional fossil fuels aggravates the energy shortage and environmental pollution problems, and the development of green renewable energy has become an important issue to be solved. Among various renewable resources, lignocellulosic biomass (LCB) is of great concern due to its wide source, low cost and no direct competition with food security [1] . LCB mainly comes from crop straw, forestry waste and other organic solid waste, and can release abundant fermentable sugars after pretreatment and hydrolysis, providing carbon source for microbial fermentation [2] . However, its hydrolysate composition is complex, usually containing six-carbon sugars (mainly glucose), five-carbon sugars (mainly xylose) and inhibitors such as acetic acid, furfural and phenols [3] , which significantly increases the difficulty of efficient utilization by downstream microorganisms. Saccharomyces cerevisiae (S. cerevisiae) Saccharomyces cerevisiae has natural high ethanol production capacity, good industrial adaptability and clear genetic background, and is considered to be one of the most potential production platforms for lignocellulosic biofuel ethanol [4,5] . However, compared with glucose, S. cerevisiae itself lacks the ability to directly and efficiently utilize xylose, and xylose accounts for an important proportion in hemicellulose hydrolysate. Therefore, how to realize the efficient co-fermentation of glucose and xylose in a complex mixed sugar environment is one of the core problems in constructing a lignocellulosic biofuel production platform.

[0003] At present, the ability of S. cerevisiae to utilize xylose as a substrate is mainly achieved by introducing an exogenous heterologous xylose metabolic pathway such as xylose reductase-xylose dehydrogenase (XR-XDH) pathway or xylose isomerase (XI) pathway, and certain modification of metabolic network targets such as pentose phosphate pathway, which can realize the full utilization of xylose by S. cerevisiae [6,7] . However, under real lignocellulosic hydrolysate and industrial conditions, due to factors such as complex substrate, inhibitor accumulation and stress response, the performance of the obtained xylose utilization yeast strain is limited, and even in some lignocellulosic hydrolysate containing more inhibitors, the strain cannot grow normally. Therefore, it is urgent to construct a S. cerevisiae for high-efficiency utilization of cellulose hydrolysate.

[0004] Saccharomyces cerevisiae has two common life styles, haploid and diploid, which can be converted between each other. Through mating, two haploid spores fuse to produce diploid; while diploid can produce haploid spores through meiosis under certain conditions, and the occurrence of these changes is determined by the mating type of the strain. The mating type of S. cerevisiae is controlled by the MAT locus located on the right arm of chromosome III, and there are two alleles on the MAT locus MATa and MATa , which determine whether the mating type of the yeast is a or α [8] . The yeast mating type conversion model was proposed by Herskowitz I et al [9] . The mutual conversion of yeast mating types MATa and MATa is caused by the product of HO gene. HO The gene is located on chromosome IV and encodes the Ho protein of site-specific restriction endonuclease. Ho protein is one of the members of the endonuclease family for intragenic sequence transposition. S. cerevisiae changes from MATa to MATa or from MATa to MATa , and this process includes the replacement of the gene information on the MAT locus by the replication information of a silent gene in HMLa or HMLa . In addition, the conversion of mating type can also be achieved by means of CRISPR plasmid

[10] .

[0005] References [1] Isikgor, Furkan H., and C. Remzi Becer. "Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers." Polymer chemistry 6.25 (2015): 4497-4559. [2] Wang, L., Bilal, M., Tan, C. et al. Industrialization progress of lignocellulosic ethanol. Syst Microbiol and Biomanuf 2, 246-258 (2022). [3]Moysés, Danuza Nogueira et al. “Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects.” International journal ofmolecular sciences vol. 17,3 207. 25 Feb. 2016, doi:10.3390 / ijms17030207. [4]Kwak, Suryang et al. “Production of biofuels and chemicals from xylose using native and engineered yeast strains.” Biotechnology advancesvol. 37,2 (2019): 271-283. doi:10.1016 / j.biotechadv.2018.12.003. [5]Li, Xiaowei et al. “Harnessing xylose pathways for biofuelsproduction.” Current opinion in biotechnology vol. 57 (2019): 56-65. doi:10.1016 / j.copbio.2019.01.006. [6]Liu, Ruo-Ying et al. “Multiscale metabolic engineering inbiological lignin valorization.” Innovation (Cambridge (Mass.) vol. 6,11100993. 13 Jun. 2025, doi:10.1016 / j.xinn.2025.100993. [7] Institute of Synthetic Biology, Tianjin University. Saccharomyces cerevisiae using xylose: 202410401158.3 [P]. 2025-10-14.

[0006] [8]Sprague, GF Jr et al. “Control of yeast cell type by the matingtypelocus. II. Genetic interactions between MAT alpha and unlinked alpha-specific STE genes.” Journal of molecular biology vol. 153,2 (1981): 323-35.doi:10.1016 / 0022-2836(81)90281-3. [9]GuthrieC, Fink GR. Guideto yeast genetics and molecular and cellbiology. Methods in enzymology. Part A. Elesevier academic press NK, 2004.194: 277-278.

[10] Tianjin University. A method for converting yeast mating types: 201610102424.8[P]. 2019-08-06. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a brewing yeast that efficiently utilizes cellulose hydrolysate.

[0008] The second objective of this invention is to provide a method for constructing the above-mentioned brewing yeast that efficiently utilizes cellulose hydrolysate.

[0009] A third objective of this invention is to provide an application of the above-mentioned brewing yeast that efficiently utilizes cellulose hydrolysate.

[0010] The technical solution of this invention is summarized as follows: A method for constructing a brewing yeast that efficiently utilizes cellulose hydrolysate includes the following steps: 1) Select haploid chassis strains of *Saccharomyces cerevisiae* that metabolize xylose via the xylose isomerase pathway; 2) The industrial ethanol yeast was desporinated to obtain haploid industrial yeast strains; 3) The strains obtained in step 1) are fused with the strains obtained in step 2), and screened to obtain a brewing yeast that efficiently utilizes cellulose hydrolysate.

[0011] Preferably, the xylose isomerase pathway xylose metabolism Saccharomyces cerevisiae haploid chassis strain is Saccharomyces cerevisiae SyBE006.

[0012] Preferably, the industrial ethanol-producing yeast is Angel yeast, Pichia pastoris, or saccharifying yeast.

[0013] The above method was used to construct a brewing yeast that efficiently utilizes cellulose hydrolysate.

[0014] The above-mentioned application of brewing yeast to efficiently produce ethanol from cellulose hydrolysate.

[0015] Beneficial effects This invention relates to a highly efficient brewing yeast that utilizes cellulose hydrolysate. In shake-flask mode, it can completely consume 70 g / L of glucose in acid-treated cellulose hydrolysate within approximately 24 hours, while simultaneously beginning to gradually consume xylose. Within 72 hours, it can completely consume 20 g / L of xylose in the acid-treated cellulose hydrolysate. Growth rate OD 600 The maximum value can reach around 15.0, producing 45 g / L of ethanol under aerobic conditions. This achieves the goal of rapid growth in industrial cellulose hydrolysate and full utilization of glucose and xylose as carbon sources for growth, further generating ethanol. Attached Figure Description

[0016] Figure 1 Comparison of growth performance of haploid chassis strains of Saccharomyces cerevisiae with different xylose metabolism pathways (SyBE006, SyBE007 and yGS001) in YPX medium.

[0017] Figure 2 The growth of haploid Angel yeast and SyBE006 obtained from sporulation in alkaline-treated industrial cellulose hydrolysate.

[0018] Figure 3 The growth of diploid fusion strains in alkaline-treated hydrolysate is shown, with A representing OD. 600 B represents the sugar consumption situation.

[0019] Figure 4 To compare the growth of the screened A15XM fusion strain with the diploid SyBE006DIP constructed by SyBE006 itself in acid-treated cellulose hydrolysate.

[0020] Figure 5 The fermentation results of fusion strain A15XM and fusion strain SKL001 used for replication experiments in acid-treated cellulose hydrolysate are shown. A represents OD600 growth and B represents sugar consumption. Detailed Implementation

[0021] brewing yeast Saccharomyces cerevisiaeSyBE006 (abbreviated as SyBE006) is SyBE003 in the authorized patent CN201210507118.4 "A Saccharomyces cerevisiae strain and its use in the co-fermentation of glucose and xylose to produce ethanol". It has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCCNo.6633. brewing yeast Saccharomyces cerevisiae SyBE007 (abbreviated as SyBE007) is SyBE005 in the authorized patent CN201210507117.X "A recombinant yeast strain with high efficiency in metabolizing xylose and its uses". It has been deposited at the China General Microbiological Culture Collection Center, with the accession number CGMCC No. 6634.

[0022] brewing yeast Saccharomyces cerevisiae yGS001 was deposited on March 8, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29981.

[0023] Table 1 Information on other strains used in this invention

[0024] YPX medium consists of: xylose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, and the remainder is water.

[0025] YPKAc prespore liquid culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 6.7 g / L yeast-free nitrogen source, 10 g / L potassium acetate, 10.2 g / L potassium hydrogen phthalate, with the balance being water, and the pH adjusted to 5.5 with 10 mol / L potassium hydroxide.

[0026] The high-efficiency liquid sporulation medium consists of: 10 g / L potassium acetate, 0.2 g / L raffinose, and the remainder is water.

[0027] Preparation method of alkali-treated cellulose hydrolysate: 1) Mix 1 g of cut corn stalks with 0.2 ml of 2 mol / L NaOH aqueous solution, stir well, and let stand for 120 h; 2) Remove the material and dry it at 160℃; 3) Enzymatic hydrolysis was performed using cellulase Accellaease 1500 and hemicellulase Multifect Xylanase. The ratio of the material obtained in step 2) to the two enzymes was 1 g: 0.08 ml: 0.04 ml. The pH was adjusted to 4.8 using concentrated sulfuric acid. 4) The enzymatic hydrolysis reaction was carried out in a shaker at 50℃ and 200 rpm for 72 h to obtain an alkaline-treated cellulose hydrolysate.

[0028] The main components of alkali-treated cellulose hydrolysate are: 77 g / L glucose; 50 g / L xylose; 2.1 g / L acetic acid; 0.2 g / L lactic acid; and 0.1 g / L glycerol (the specific content of the components may fluctuate due to storage time and detection errors).

[0029] Preparation method of acid-treated cellulose hydrolysate: 1) Corn stalk crushing; 2) Pre-soak in 1% dilute sulfuric acid for 12 hours, with a solid-liquid ratio of 1 g: 10 ml; 3) Treat with 1.8 MPa steam for 8 minutes to induce steam explosion; 4) Enzymatic hydrolysis was performed using cellulase Accellaease 1500 and hemicellulase Multifect Xylanase. The ratio of the material obtained in step 3) to the two enzymes was 1 g: 0.08 ml: 0.04 ml, and citrate buffer with pH=4.8 was added. 5) The enzymatic hydrolysis reaction was carried out in a shaker at 50℃ and 200 rpm for 72 h to obtain acid-treated cellulose hydrolysate.

[0030] The main components of acid-treated cellulose hydrolysate are: 2.2 g / L cellobiose; 70 g / L glucose; 20 g / L xylose; 3.2 g / L arabinose; 1.3 g / L lactic acid; 0.3 g / L glycerol; and 6.7 g / L acetic acid (the specific content of the components may fluctuate due to storage time and detection errors).

[0031] Methods for detecting glucose metabolism: High performance liquid chromatography (HPLC). The high-pressure pump was a Waters 1515, the column was an Aminex HPX-87H (BioRad, CA, USA), the column temperature was controlled at 65℃, the detector was a Waters 2414 differential detector, the temperature was controlled at 40℃, the mobile phase was 5mM sulfuric acid, and the flow rate was controlled at 0.6mL / min.

[0032] The present invention will be further described below through specific embodiments.

[0033] Example 1 A method for constructing a brewing yeast that efficiently utilizes cellulose hydrolysate includes the following steps: 1. Add 50 ml of YPX medium to a shake flask and compare different xylose-metabolizing Saccharomyces cerevisiae haploid chassis strains. Based on... Figure 1As a result, the haploid chassis strain of Saccharomyces cerevisiae that metabolizes xylose via the xylose isomerase pathway, Saccharomyces cerevisiae SyBE006, was selected. 2. The industrial-grade ethanol yeast Angel yeast was desporinated to obtain a haploid industrial yeast strain, namely haploid Angel yeast; The methods for spore formation and despore removal are as follows: 1) First, the Angel yeast was cultured on YPD solid medium (known) at 30℃ for 2 days to activate the strain; 2) Pick an appropriate amount of colonies from the activated plate and inoculate them into 3 mL of YPD liquid medium, and culture them simultaneously at 30°C until the exponential growth phase; 3) Take an appropriate amount of synchronous culture into a 1.5 mL sterile EP tube, centrifuge at 4000 rpm for 1 min, wash 3 times with sterile water; then inoculate into 3 mL YPKAc pre-spore liquid medium with OD600 = 0.5, and incubate at 25℃ and 220 rpm for 24 h. 4) Take 1.5 mL of the culture obtained in step 3), centrifuge at 5000 rpm for 3 min, discard the supernatant, collect the bacterial cells, wash three times with sterile water, and finally resuspend the bacterial cells in 3 mL of high-efficiency liquid sporulation medium with OD600 = 0.5, and incubate at 25℃ and 220 rpm for 3 days. 5) Microscopic examination: Spot 10 μL of the bacterial solution from step 4) onto a glass slide and observe the formation of spores (tetrads) under a microscope; 6) Take 50 μL of the bacterial culture from step 4), add 4 μL (adjust according to enzyme activity) of cell wall disrupting enzyme (Zymolyase), digest at 37℃ for 30 min, and dilute with 200 μL of ddH2O; 7) Pipette 20 μL of the diluent from step 6) onto the streak line of a YPD plate (prepared at least one week ago), stand the plate upright to allow the cell sap to form strips, let it absorb, and then use a desporer to separate the cells. 8) After verifying the mating type of spores, alkali-treated cellulose hydrolysate was used for high-throughput initial screening in 24-well plates. 2 ml of alkali-treated cellulose hydrolysate was added to each well of the 24-well plate, with an initial inoculum size of OD0.05. 600 =0.2, cultured at 30℃ and 220 rpm. Screening results are shown below. Figure 2 , to obtain OD 600 Higher levels of haploid Angel yeast A15, A17, A18, A26, A29, etc.

[0034] 9) PCR verification method for the mating type of Saccharomyces cerevisiae: PCR amplification was performed using the following three primers.

[0035] MAT-F (a sequence located to the right of the MAT locus, pointing to the MAT locus); MAT-a (a-specific DNA sequence located on MATa and HMRa); MAT-α (an α-specific DNA sequence located on MATα and HMRα); The PCR products were detected by gel electrophoresis. The PCR product of type a yeast cells was 544 bp, the PCR product of type α yeast cells was 404 bp, and the PCR products of diploid yeast cells were two: 404 bp and 544 bp.

[0036] The mating type of the yeast strain was determined based on the length of the PCR product. The primer sequences used are as follows: Table 2 Primers used for mating-type PCR validation

[0037] 3. Construct a brewing yeast that efficiently utilizes cellulose hydrolysate.

[0038] SyBE006 was fused with haploid Angel yeast strains A15, A17, A18, A26, and A29, respectively. The fusion method for reverse mating type haploids is as follows: 1) Pipette 30 μL of sterile water into a 1.5 mL sterile centrifuge tube, and pick up equal amounts of SyBE006 and haploid Angel yeast into centrifuge tubes respectively, and mix them evenly; 2) Take 20 μL of the bacterial culture from step 1) and drop it onto a YPD plate, then incubate it in a 30℃ incubator for 1 day; 3) Microscopic examination: Pick yeast cells from the YPD plate in step 2) and place them in 200 μL of sterile water. Take 10 μL and spot it on a glass slide. Observe under a microscope that the cells have fused, that is, dumbbell-shaped and raised cells. 4) Dilute the bacterial suspension from step 3) 10 times with sterile water to obtain a diluted solution. Pipette 20 μL onto the streak line of a YPD plate that has been prepared for more than one week. Stand the plate upright so that the diluted solution forms a strip and allow it to absorb. Use a spore remover to pick out diploid cells (cells that are currently merging). Incubate at 30°C for 2 days.

[0039] 5) After verification of fusion cell mating types, alkali-treated cellulose hydrolysate was used for high-throughput screening in 24-well plates. 2 ml of alkali-treated cellulose hydrolysate was added to each well of the 24-well plate, with an initial inoculum size of OD0.05. 600 =0.2, cultured at 30℃ and 220 rpm. The screening results of the obtained diploid strains are shown below. Figure 3 Among them, strain A15XM performed better. This strain was obtained by fusing SyBE006 with A15.

[0040] 4. Validation of brewing yeast fermentation using cellulose hydrolysate efficiently.

[0041] SyBE006 was cross-type converted using CRISPR / CAS9 technology, and self-fusion was performed to construct diploid SyBE006DIP. Fermentation was then carried out in acid-treated cellulose hydrolysate. Figure 4 The SyBE006DIP sample failed to grow normally, indicating that the *Saccharomyces cerevisiae* A15XM strain constructed using the cellulose hydrolysate by the method of this invention has stronger tolerance while retaining the characteristic of co-utilizing glucose and xylose. SyBE006 was fused with A15 to construct SKL001, which served as the experimental verification target for the replication of A15XM. Fermentation results are shown in […]. Figure 5 .

[0042] This invention relates to a highly efficient brewing yeast that utilizes cellulose hydrolysate. In shake-flask mode, it can completely consume 70 g / L of glucose in the acid-treated cellulose hydrolysate to generate ethanol in approximately 24 hours, while simultaneously beginning to gradually consume xylose. By 72 hours, it can completely consume 20 g / L of xylose in the acid-treated cellulose hydrolysate. (Growth rate OD) 600 The maximum value can reach around 15.0, producing 45 g / L of ethanol under aerobic conditions. This achieves the goal of rapid growth in industrial cellulose hydrolysate and full utilization of glucose and xylose as carbon sources for growth, further generating ethanol.

[0043] Experiments have shown that replacing Angel yeast in Example 1 with Pichia kudriozweeni or Saccharomyces cerevisiae, while keeping other aspects the same as in Example 1, yields brewing yeasts that efficiently utilize cellulose hydrolysate.

Claims

1. A method for constructing brewing yeast that efficiently utilizes cellulose hydrolysate, characterized by: Includes the following steps: 1) Select haploid chassis strains of *Saccharomyces cerevisiae* that metabolize xylose via the xylose isomerase pathway; 2) The industrial ethanol yeast was desporinated to obtain haploid industrial yeast strains; 3) The strains obtained in step 1) are fused with the strains obtained in step 2), and screened to obtain a brewing yeast that efficiently utilizes cellulose hydrolysate.

2. The method according to claim 1, characterized in that: The xylose isomerase pathway involved in xylose metabolism in the *Saccharomyces cerevisiae* haploid chassis strain is *Saccharomyces cerevisiae* SyBE006.

3. The method according to claim 1, characterized in that: The industrial ethanol-producing yeast is Angel Yeast, Pichia pastoris, or Saccharifying yeast.

4. A brewing yeast that efficiently utilizes cellulose hydrolysate, constructed by the method of any one of claims 1-3.

5. The application of the brewing yeast of claim 4 to efficiently produce ethanol from cellulose hydrolysate.

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