Saccharomyces cerevisiae strain, construction method, application and compound strain

Abstract

The invention discloses a saccharomyces cerevisiae strain, a construction method, application and a compound strain, the preservation number of the saccharomyces cerevisiae strain SEB26 is CGMCC No.33178, the preservation date is December 24, 2024, the preservation unit is China General Microbiological Culture Collection Center, and the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The SEB26 strain disclosed by the invention can simultaneously display beta-glucan endonuclease and beta-glucan exonuclease, expands a substrate spectrum of SEB5, endows the strain with glucan metabolism capability, and endows the strain with utilization of oligosaccharide in straw hydrolysate, so that a recombinant strain capable of directly utilizing oligosaccharide for fermentation is developed, the total sugar utilization rate is increased, and the production cost is reduced. Furthermore, the treatment cost of a concentrated acid hydrolysis process is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of Saccharomyces cerevisiae strains, and specifically relates to Saccharomyces cerevisiae strains, construction methods and their applications, as well as a composite strain. Background Art

[0002] Using lignocellulosic biomass such as crop straw to produce fuel ethanol can not only realize the resource utilization of organic solid waste, but also effectively alleviate energy shortage and environmental crisis. Saccharomyces cerevisiae, as a traditional ethanol-producing strain, has become an ideal choice for cellulose fuel ethanol production due to its excellent biosafety, strong stress resistance and easy gene operation.

[0003] Lignocellulose is mainly composed of tightly intertwined cellulose, hemicellulose and lignin, forming a complex network structure, which makes it difficult to be biodegradable. In order to reduce its crystallinity and improve accessibility, it is usually necessary to destroy its complex structure through pretreatment to facilitate the subsequent enzymatic hydrolysis process. Common pretreatment methods include steam explosion, acid-base treatment, etc. After pretreatment, under the synergistic action of cellulase and hemicellulase, cellulose and hemicellulose are hydrolyzed into monosaccharides such as glucose and xylose. These monosaccharides are then fermented into ethanol by Saccharomyces cerevisiae. Compared with the "pretreatment + enzymatic hydrolysis" process, the concentrated acid hydrolysis process has the advantages of low energy consumption, low toxic by-products and no need for cellulase input, thus reducing the production cost. However, the concentrated acid hydrolysis sugar solution usually contains a certain amount of oligosaccharides, which need to be further hydrolyzed by high-temperature dilute acid to be completely converted into monosaccharides, which undoubtedly increases the treatment cost.

[0004] We previously successfully constructed a Saccharomyces cerevisiae strain (SEB5, deposit number CGMCC11325) that can simultaneously utilize glucose, xylose and cellobiose for ethanol fermentation. However, this strain cannot utilize oligosaccharides for fermentation, which limits its application in the concentrated acid hydrolysis process.

[0005] In view of this, this patent application is proposed. Summary of the Invention

[0006] To solve the problem of oligosaccharide utilization, the present invention provides the Saccharomyces cerevisiae strain SEB26, its construction method and application, and also provides a composite strain containing Saccharomyces cerevisiae strain SEB25 and Saccharomyces cerevisiae strain SEB26.

[0007] Specifically, the following technical solutions are adopted to achieve:

[0008] The first object of the present invention is to provide a Saccharomyces cerevisiae strain, which is Saccharomyces cerevisiae SEB26, with a deposit number of CGMCC No. 33178, a deposit date of December 24, 2024, and a deposit unit of the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The second object of the present invention is to provide a construction method of the above-mentioned Saccharomyces cerevisiae strain SEB26, which includes the following steps:

[0010] Step 1, construct strain SEB5EG:

[0011] (1) Construct pMEL13-PHO13 plasmid and β-glucan endonuclease EG2 surface display gene cassette;

[0012] (2) Introduce Cas9-NAT plasmid into Saccharomyces cerevisiae strain SEB5 to obtain strain SEB5-Cas9;

[0013] (3) Introduce pMEL13-PHO13 plasmid and β-glucan endonuclease EG2 surface display gene cassette into strain SEB5-Cas9;

[0014] (4) Remove the Cas9-NAT plasmid and pMEL13-PHO13 plasmid of the correct transformant to obtain strain SEB5 EG;

[0015] Step 2, construct strain SEB26:

[0016] (5) Construct pMEL13-ALD6 plasmid and β-glucan exohydrolase TrCBH1 surface display gene cassette;

[0017] (6) Introduce Cas9-NAT plasmid into Saccharomyces cerevisiae strain SEB5EG to obtain strain SEB5EG-Cas9;

[0018] (7) Using strain SEB5EG-Cas9 as the starting strain, transfer the β-glucan exohydrolase TrCBH1 surface display gene cassette and pMEL13-ALD6 plasmid into the cells;

[0019] (8) Remove the plasmid to obtain strain SEB26.

[0020] As a preferred design, it is characterized in that the process of constructing the β-glucan endonuclease EG2 surface display gene cassette in (1) is:

[0021] Synthesize P SED1 -S.S SED1 -TrEG2-A SED1-T SAG1 The gene was ligated to the EcoRI and SalI sites of the pUC57 vector to obtain the plasmid pUC57-SED1-TrEG2. P SED1 、S.S SED1 、TrEG2、A SED1 、T SAG1 The sequences of the genes are SEQ ID NO: 7, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 10, and SEQ ID NO: 11 respectively;

[0022] Using the pUC57-SED1-TrEG2 plasmid as a template, amplification was performed using primers PHO13celRE-F and PHO13celRE-R. The obtained DNA fragment is the β-glucan endonuclease EG2 surface display gene cassette. The sequences of primers PHO13celRE-F and PHO13celRE-R are SEQ ID NO: 16-17.

[0023] As a preferred design, in (3), the obtained strain was verified by colony PCR. The verification was performed using primers PHO13-dgF and PHO13-dgR. The sequences of primers PHO13-dgF and PHO13-dgR are SEQ ID NO: 18-19.

[0024] As a preferred design, the process of constructing the pMEL13-ALD6 plasmid in (5) is as follows:

[0025] Construct a double-stranded gRNA fragment: Design a gRNA recognition sequence within the coding sequence of the ALD6 gene. Synthesize a primer pair ALD6 TG F / R with a 20bp recognition sequence and 50bp homologous arms upstream and downstream. Dilute, mix, heat, and cool the two single-stranded primers at room temperature in sequence;

[0026] Amplify the linear backbone of the gRNA plasmid using the pMEL13 plasmid as a template;

[0027] Recombinantly ligate the double-stranded gRNA fragment and the pMEL13 linear backbone to obtain the pMEL13-ALD6 plasmid;

[0028] The sequence of the primer pair ALD6 TG F / R is SEQ ID NO: 1-2;

[0029] During the process of amplifying the linear backbone of the gRNA plasmid, the primer sequences of the plasmid backbone are shown as SEQ ID NO: 3-4.

[0030] As a preferred design, the process of constructing the β-glucan exohydrolase TrCBH1 surface display gene cassette in (5) is as follows:

[0031] Synthesis of P SED1 -S.S SED1 -TrCBH1-A SED1 -T SAG1 The gene was ligated to the EcoRI and SalI sites of the pUC57 vector to obtain the pUC57-SED1-TrCBH1 plasmid. Using the pUC57-SED1-TrCBH1 plasmid as a template, amplification was performed with primers ALD6celRE-F and ALD6 celRE-R, and the obtained DNA fragment was the β-glucan exohydrolase TrCBH1 surface display gene cassette;

[0032] The sequences of the TrCBH1 gene were SEQ ID NO: 22;

[0033] The sequences of primers ALD6 celRE-F and ALD6 celRE-R were SEQ ID NO: 5-6.

[0034] As a preferred design, the obtained strain was verified by colony PCR in (7). The verification was performed using primers ALD6-dgF and ALD6-dgR, and the sequences of primers ALD6-dgF and ALD6-dgR were SEQ ID NO: 12-13.

[0035] The third object of the present invention is to provide the application of the Saccharomyces cerevisiae strain SEB26 obtained by any of the above in the fermentation production of ethanol using oligosaccharides.

[0036] The fourth object of the present invention is to provide a composite strain, including the Saccharomyces cerevisiae SEB26 strain as described above or the Saccharomyces cerevisiae SEB26 strain constructed by any of the above methods, and further including the Saccharomyces cerevisiae SEB25 strain, and the preservation number of the Saccharomyces cerevisiae SEB25 strain is CGMCC No. 33177.

[0037] As a preferred design, the mixing ratio of the SEB25 strain to the SEB26 strain is 1:1-7:3.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] 1. The strain SEB26 obtained in the embodiments of the present invention can simultaneously display endo-β-glucanase and exo-β-glucanase, expanding the substrate spectrum of SEB5, endowing the strain with the ability to metabolize glucan, endowing it with the utilization of oligosaccharides in the straw hydrolysis liquor, and thus developing a recombinant strain that can directly utilize oligosaccharides for fermentation, which is one of the effective means to improve the total sugar utilization rate and reduce the treatment cost of the concentrated acid hydrolysis process.

[0040] 2. In order to solve the problem that in the current concentrated acid hydrolysis process, oligosaccharides need to be further hydrolyzed with dilute acid at high temperature, resulting in a complex treatment process and high cost, specific hemicellulases (xylanase and xylosidase) and cellulases (endo-β-glucanase and exo-β-glucanase) were respectively expressed on the cell surface of the SEB5 strain, and these two recombinant strains were combined and fermented with the straw concentrated acid hydrolysis liquor containing oligosaccharides. The results showed that this combination strategy could significantly increase the ethanol yield. The strains constructed in this study can also be applied to the ethanol fermentation in the "pretreatment + enzymatic hydrolysis" process, reducing the input of cellulase. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts. In the drawings:

[0042] Figure 1 For the glucan fermentation results in the examples, a represents the concentrations of glucan and glucose; b represents the dry cell weight and ethanol concentration.

[0043] Figure 2 For the xylan fermentation results in the examples, a represents the dry cell weight (DCW) and xylose concentration; b represents the ethanol and xylitol concentrations.

[0044] Figure 3 For the fermentation results of the straw concentrated acid hydrolysis liquor in the examples, a represents the concentrations of glucose, xylose and cellobiose; b represents the dry cell weight, ethanol and xylitol concentrations.

[0045] Figure 4 It is a comparison chart of the fermentation performance of the cell surface-displaying cellulase strains of the disclosed technology and this patent application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0046] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to embodiments and the accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and do not limit the present invention.

[0047] Embodiment:

[0048] The present invention constructs the Saccharomyces cerevisiae strain SEB26 and also constructs the Saccharomyces cerevisiae strain SEB25. The surface of the Saccharomyces cerevisiae strain SEB25 can display hemicellulases (xylanase and xylosidase) to effectively utilize xylan, and the surface of the Saccharomyces cerevisiae strain SEB26 can display cellulases (endo-β-glucanase and exo-β-glucanase). At the same time, these two recombinant strains are combined to form a composite strain, which can ferment the straw concentrated acid hydrolysis solution containing oligosaccharides, significantly increasing the ethanol yield.

[0049] I. Preservation Information

[0050] The preservation information of the Saccharomyces cerevisiae strain SEB25 is as follows:

[0051] Preservation number: CGMCC No. 33177, preservation date: December 24, 2024, preservation unit: China General Microbiological Culture Collection Center, preservation unit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0052] The preservation information of the Saccharomyces cerevisiae strain SEB26 is as follows:

[0053] Preservation number: CGMCC No. 33178, preservation date: December 24, 2024, preservation unit: China General Microbiological Culture Collection Center, preservation unit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0054] II. Construction process of the Saccharomyces cerevisiae strain SEB25 and the Saccharomyces cerevisiae strain SEB26.

[0055] SEB25 is constructed by the following method:

[0056] 1. The starting strain is SEB5, which is preserved in the China General Microbiological Culture Collection Center with the preservation number CGMCC11325.

[0057] 2. Medium

[0058] The culture media used are shown in Table 1. If it is a solid culture medium, 2% agar powder is added before sterilization. The sterilization conditions are 0.1 MPa, 121 °C, and 15 min. After the culture medium is cooled to 50 - 60 °C, antibiotics are added.

[0059] Table 1 Composition of the culture media used

[0060]

[0061] 3. Plasmids, strains, and primers

[0062] The plasmids and strains used in the construction process are shown in Table 2; the primers for constructing the gRNA plasmid are shown in Table 3; the primers for strain transformation are shown in Table 4.

[0063] Table 2 Plasmid and strain information

[0064]

[0065]

[0066] Note: P: Promoter; S.S: Signal Sequence; A: Anchor; T: Terminator.

[0067] Table 3 Primers for constructing the gRNA plasmid

[0068]

[0069] Note: F: Forward primer; R: Reverse primer; Underline: 20 bp recognition sequence.

[0070] Table 4 Primers required for strain transformation

[0071]

[0072]

[0073] Note: RE: Repair fragment; dg: Verification primer; F: Forward primer; R: Reverse primer

[0074] 4. Construction of strain SEB5AD

[0075] 4.1 Construction of the gRNA plasmid pMEL13-ALD6

[0076] 1) Construction of the double-stranded gRNA fragment

[0077] Using the genomic information of the model yeast S288c as a reference, the gRNA recognition sequence (20bp) was designed within the coding sequence of the ALD6 gene using the CHOPCHOP website. The primer pair ALD6 TG F / R with a 20bp recognition sequence and 50bp homologous arms upstream and downstream was synthesized (the sequences are shown in Table 3). The two single-stranded primers were diluted to 10 μM with sterile water respectively, mixed in equal volumes, heated at 95 °C for 5 min, and cooled to room temperature to obtain the double-stranded gRNA fragment.

[0078] 2) Amplify the linear backbone of the gRNA plasmid

[0079] The linear backbone was amplified using the pMEL13 plasmid as a template. The PCR reaction system and reaction conditions are shown in Table 5 below. The linear backbone of the gRNA plasmid was obtained after purifying the PCR product.

[0080] Table 5 PCR amplification of the gRNA linear backbone

[0081]

[0082]

[0083] The template plasmid was digested with FastDigest Dpn I. The digestion system and reaction conditions are shown in Table 6 below. The amount of DpnI used is determined by the plasmid amount (1 μg plasmid plus 1 μL DpnI). The gRNA linear backbone was obtained after purification.

[0084] Table 6 Digestion system of the gRNA linear backbone template

[0085]

[0086] 3) Gibson ligation of the gRNA fragment and the linear backbone

[0087] The double-stranded gRNA fragment obtained in 1) and the pMEL13 linear backbone obtained in 2) were ligated by homologous recombination. The Gibson ligation reaction system and conditions are shown in Table 7 below. Take 5 μL of the ligation solution and transform it into Escherichia coli. The bacterial solution was spread on an LB+Kan plate and cultured overnight at 37 °C. The transformants were inoculated into a test tube containing 5 mL of LB+Kan liquid medium and cultured for 12 - 16 h (160 rpm, 37 °C). The bacterial cells were collected to extract the gRNA plasmid, and sequencing was performed to confirm the correct gRNA plasmid pMEL13-ALD6 was obtained.

[0088] Table 7 Gibson ligation reaction system

[0089]

[0090] 4.2 Construction of the repair fragment - xylanase xlnD surface display gene cassette

[0091] Commission a gene synthesis company to synthesize P SED1 -S.S AnxlnD -AnxlnD-A SED1 -T SAG1 genes and ligate them to the EcoRI and SalI sites of the pUC57 vector. Name the recombinant plasmid pUC57-SED1-AnxlnD. Among them, the AnxlnD gene is derived from Aspergillus niger (commissioned a gene synthesis company to synthesize), and codon optimization was performed during synthesis. The base sequence is shown in Table 8 below. Using the pUC57-SED1-AnxlnD plasmid as a template, amplify with primers ALD6celRE-F and ALD6celRE-R (primer sequences are shown in Table 4 above). The obtained DNA fragment is the xylanase xlnD surface display gene cassette (with ALD6 homologous arms on both sides). The PCR reaction system and reaction conditions are shown in Table 9. The PCR product is purified and used for transformation.

[0092] Table 8 Xylanase xlnD surface display gene cassette sequence

[0093]

[0094]

[0095]

[0096]

[0097] Table 9 PCR amplification repair fragment

[0098]

[0099] 4.3 Preparation of Cas9-NAT plasmid

[0100] Inoculate Escherichia coli containing the Cas9-NAT plasmid into 5 mL of LB+NAT liquid medium and culture it with shaking at 37 °C and 160 rpm for 12 - 16 h. Centrifuge to collect the bacteria and extract the Cas9-NAT plasmid.

[0101] 4.4 CRISPR / Cas9 transformation

[0102] (1) Introduce the Cas9-NAT plasmid into the Saccharomyces cerevisiae strain by the lithium acetate transformation method

[0103] a. Streak the strain SEB5 on a 2% YPD plate for activation. Take an appropriate amount of bacteria and inoculate them into 5 mL of 2% YPD liquid medium. Culture with shaking at 30 °C and 160 rpm for 16 h;

[0104] b. Inoculate 2 mL of the bacterial solution into 100 mL of 2% YPD culture medium and culture at 30 °C and 160 rpm for 2 - 3 h. When OD 600 reaches 0.2 - 0.3, centrifuge to collect all the bacteria. Wash the bacterial pellet twice with sterile water and then resuspend it in 300 μL of sterile water, and place it on ice for later use;

[0105] c. Boil salmon sperm DNA (ssDNA) at 100 °C for 5 min and place it on ice for later use;

[0106] d. Sequentially add 50% PEG4000 (115 μL), 4 M lithium acetate solution (5 μL), ssDNA (10 μL), Cas9 - NAT plasmid (100 ng), and yeast cells (50 μL) into a 1.5 mL centrifuge tube. After mixing, perform heat shock at 42 °C for 40 min;

[0107] e. Centrifuge at 8,000×g for 1 min, discard the supernatant, wash the cells twice with sterile water, add 1 mL of 2% YPD culture medium, and culture at 30 °C and 160 rpm for 2 - 3 h;

[0108] f. Centrifuge at 8,000×g for 1 min, discard the supernatant, and resuspend the cells with 1 mL of sterile water. Take 100 μL of the bacterial solution and spread it on a 2% YPD + NAT plate, and culture at 30 °C for 2 - 3 d;

[0109] g. Streak the transformants on a 2% YPD + NAT plate. The transformants that can grow contain the Cas9 - NAT plasmid.

[0110] (2) Knock - in the xylanase xlnD surface display gene cassette at the ALD6 locus

[0111] a. Streak the strain SEB5 - Cas9 containing the Cas9 - NAT plasmid on a 2% YPD + NAT plate for activation. Take an appropriate amount of bacteria and inoculate it into 5 mL of 2% YPD + NAT liquid medium, and culture with shaking at 30 °C and 160 rpm for 16 h;

[0112] b. Inoculate 2 mL of the bacterial solution into 100 mL of 2% YPD + NAT culture medium and culture at 30 °C and 160 rpm for 2 - 3 h. When OD 600 reaches 0.2 - 0.3, centrifuge to collect all the bacteria. Wash the bacterial pellet twice with sterile water and then resuspend it in 300 μL of sterile water, and place it on ice for later use;

[0113] c. Boil salmon sperm DNA (ssDNA) at 100 °C for 5 min and place it on ice for later use;

[0114] d. In a 1.5 mL centrifuge tube, add 50% PEG4000 (240 μL), 4 M lithium acetate solution (9 μL), ssDNA (25 μL), pMEL13-ALD6 plasmid (600 ng), and xlnD repair fragment (1.5 - 2.5 μg) in sequence. Make up to 351 μL with sterile water, add yeast cells (50 μL), mix well, and heat shock at 42 °C for 40 min;

[0115] e. Centrifuge at 8,000 × g for 1 min, discard the supernatant, wash the cells twice with sterile water, add 1 mL of 2% YPD culture medium, and culture at 30 °C and 160 rpm for 2 - 3 h;

[0116] f. Centrifuge at 8,000 × g for 1 min, discard the supernatant, resuspend the cells with 150 μL of sterile water, spread all the bacterial solution on a 2% YPD + NAT + G418 plate, culture at 30 °C for 2 - 3 d, and pick the transformants for colony PCR verification.

[0117] 4.5 Colony PCR verification

[0118] a. In a 1.5 mL centrifuge tube, add 95 μL of 1% SDS, 5 μL of 4 M lithium acetate solution, and an appropriate amount of bacteria, vortex, and incubate at 75 °C for 10 min;

[0119] b. Add 300 μL of absolute ethanol to the centrifuge tube, vortex; centrifuge at 13,000 rpm at room temperature for 3 min, discard all the supernatant, and dry with the lid open at 37 °C for 10 min;

[0120] c. Add 100 μL of sterile water, vortex, centrifuge at 13,000 rpm at room temperature for 1 min, and retain the supernatant;

[0121] d. Measure the concentration of the supernatant, adjust the OD to 10 - 20 ng / μL, take 1 μL as the template, and perform PCR verification using primers ALD6-dgF and ALD6-dgR. The reaction system and reaction conditions are shown in Table 10;

[0122] e. Verify the target band by 1% agarose gel electrophoresis (100 V, 30 min), and sequence the correct PCR products of the band for confirmation.

[0123] Table 10 Colony PCR verification system

[0124]

[0125] 4.6 Plasmid removal

[0126] Remove the Cas9-NAT plasmid and pMEL13-ALD6 plasmid of the correctly transformed clones. The specific steps are as follows:

[0127] a. Streak the correct transformants on a 2% YPD plate, pick a small amount of cells and inoculate them into 5 mL of 5% YPD medium, and culture at 30 °C for 1 day.

[0128] b. Take 1 mL of the bacterial solution and dilute it 10 5 times. Take 100 μL of the bacterial solution and spread it on a 2% YPD plate, and culture for 1 - 2 days;

[0129] c. Resuspend a small amount of cells in 100 μL of sterile water (pick 3 single colonies for each transformant). Take 2 μL of each bacterial suspension and spot plate them on 2% YPD, 2% YPD + G418, and 2% YPD + NAT plates respectively, and culture at 30 °C for 1 - 2 days; Strains that can only grow on the 2% YPD plate have successfully removed the plasmid. Finally, the strain SEB5AD that successfully displayed xylanase on the cell surface was obtained.

[0130] 5 Construction of Strain SEB25

[0131] 5.1 Construction of the repair fragment - the xylosidase xlnB surface display gene cassette

[0132] Entrust a company to synthesize the P SED1 -S.S AnxlnB -AnxlnB-A SED1 -T SAG1 gene and ligate it to the EcoRI and SalI sites of the pUC57 vector. The recombinant plasmid was named pUC57-SED1-AnxlnB. Among them, AnxlnB is derived from Aspergillus niger, and codon optimization was performed during synthesis. The base sequence is shown in Table 11 below. Using the pUC57-SED1-AnxlnB plasmid as a template, amplify using primers PHO13celRE-F and PHO13celRE-R (primer sequences are shown in Table 4). The obtained DNA fragment is the xylosidase xlnB surface display gene cassette (with PHO13 homologous arms on both sides). The PCR reaction system and reaction conditions are the same as in Table 9. The PCR product was purified and used for transformation.

[0133] Table 11 Sequence of the xylosidase xlnB surface display gene cassette

[0134]

[0135]

[0136] Note: The sequences of other components are the same as in Table 8.

[0137] 5.2 Preparation of pMEL13-PHO13 plasmid

[0138] Inoculate Escherichia coli containing the pMEL13-PHO13 plasmid into 5 mL of LB+KAN liquid medium and culture it with shaking at 37°C and 160 rpm for 12 - 16 h. Centrifuge to collect the bacterial cells and extract the pMEL13-PHO13 plasmid.

[0139] 5.3 CRISPR / Cas9 Transformation

[0140] (1) Introduce the Cas9-NAT plasmid into the Saccharomyces cerevisiae strain by the lithium acetate transformation method

[0141] Using SEB5AD as the starting strain, the lithium acetate transformation process is the same as in 4.4(1) to obtain the SEB5AD-Cas9 strain.

[0142] (2) Knock in the xylosidase xlnB surface display gene cassette at the PHO13 locus

[0143] Using SEB5AD-Cas9 as the starting strain, transfer the xlnB surface display gene cassette P SED1 -S.S AnxlnB -AnxlnB-A SED1 -T SAG1 and the pMEL13-PHO13 plasmid into the cells. The detailed steps are the same as in 4.4(2).

[0144] 5.4 Colony PCR Verification

[0145] The steps for extracting the bacterial cell DNA are the same as in 4.5. Take 1 μL as the template and perform PCR verification using the primers PHO13-dgF and PHO13-dgR. The reaction system and reaction conditions are shown in Table 10.

[0146] 5.5 Plasmid Removal

[0147] The operation steps are the same as in 4.6, and finally, the strain SEB25 that simultaneously displays xylanase xlnD and xylosidase xlnB on the cell surface is successfully obtained.

[0148] The SEB26 strain is constructed according to the following method:

[0149] 6 Construction of Strain SEB5EG

[0150] 6.1 Construction of the Repair Fragment - β-1,3-Glucanase EG2 Surface Display Gene Cassette

[0151] Commission a company to synthesize P SED1 -S.S SED1 -TrEG2-A SED1 -T SAG1The gene was ligated to the EcoRI and SalI sites of the pUC57 vector, and the recombinant plasmid was named pUC57-SED1-TrEG2. Among them, TrEG2 was derived from Trichoderma reesei (synthesized by a gene synthesis company on commission), and codon optimization was performed during synthesis. The base sequence is shown in Table 12 below. Using the pUC57-SED1-TrEG2 plasmid as a template, amplification was carried out using primers PHO13celRE-F and PHO13celRE-R (the primer sequences are shown in Table 4), and the obtained DNA fragment is the β-glucan endonuclease EG2 surface display gene cassette (with PHO13 homologous arms on both sides). The PCR reaction system and reaction conditions are shown in Table 9. The PCR product was purified and used for transformation.

[0152] Table 12 β-glucan endonuclease EG2 surface display gene cassette sequence

[0153]

[0154]

[0155] Note: The sequences of other components are the same as those in Table 8.

[0156] 6.2 Knock-in of the β-glucan endonuclease EG2 surface display gene cassette at the PHO13 locus

[0157] Using the strain SEB5-Cas9 containing the Cas9-NAT plasmid as the starting strain, the EG2 surface display gene cassette P SED1 -S.S SED1 -TrEG2-A SED1 -T SAG1 and the pMEL13-PHO13 plasmid were transferred into the cells. The detailed steps are the same as those in 4.4(2).

[0158] 6.3 Colony PCR verification

[0159] The steps for extracting genomic DNA from the cells are the same as those in 4.5. Take 1 μL as the template and perform PCR verification using primers PHO13-dgF and PHO13-dgR. The reaction system and reaction conditions are shown in Table 10.

[0160] 6.4 Removal of the plasmid

[0161] The operation steps are the same as those in 4.6, and finally, the strain SEB5EG that displays β-glucan endonuclease EG2 on the cell surface was successfully obtained.

[0162] 7 Construction of strain SEB26

[0163] 7.1 Construction of the repair fragment - β-glucan exohydrolase TrCBH1 surface display gene cassette

[0164] Commissioned the company to synthesize P SED1 -S.S SED1 -TrCBH1-A SED1 -T SAG1 The gene was ligated to the EcoRI and SalI sites of the pUC57 vector, and the recombinant plasmid was named pUC57-SED1-TrCBH1. Among them, the TrCBH1 gene was derived from Trichoderma reesei and codon-optimized during synthesis. The base sequence is shown in Table 13 below. Using the pUC57-SED1-TrCBH1 plasmid as a template, amplification was performed using primers ALD6 celRE-F and ALD6 celRE-R (the primer sequences are shown in Table 4), and the obtained DNA fragment was the β-exoglucanase TrCBH1 surface display gene cassette (with ALD6 homologous arms on both sides). The PCR reaction system and reaction conditions are shown in Table 9. The PCR product was purified and used for transformation.

[0165] Table 13 β-exoglucanase TrCBH1 surface display gene cassette sequence

[0166]

[0167]

[0168] Note: The sequences of other components are the same as those in Table 8.

[0169] 7.2 CRISPR / Cas9 transformation

[0170] (1) The Cas9-NAT plasmid was introduced into the Saccharomyces cerevisiae strain by the lithium acetate transformation method

[0171] Using SEB5EG as the starting strain, the lithium acetate transformation process is shown in 4.4(1), and the SEB5EG-Cas9 strain was obtained.

[0172] (2) Knock in the β-exoglucanase TrCBH1 surface display gene cassette at the ALD6 locus

[0173] Using SEB5EG-Cas9 as the starting strain, the TrCBH1 surface display gene cassette P SED1 -S.S SED1 -TrCBH1-A SED1 -T SAG1 and the pMEL13-ALD6 plasmid (see the process in 4.1) were transferred into the cells, and the detailed steps were the same as those in 4.4(2).

[0174] 7.3 Colony PCR verification

[0175] The steps for extracting genomic DNA of the cells are the same as those in 4.5. Take 1 μL as the template and perform PCR verification using primers ALD6-dgF and ALD6-dgR. The reaction system and reaction conditions are shown in Table 10.

[0176] 7.4 Plasmid Removal

[0177] The operation steps are the same as those in 4.6. Finally, strain SEB26 that simultaneously displays endoglucanase EG2 and exoglucanase TrCBH1 on the cell surface is successfully obtained.

[0178] 8. Fermentation Evaluation

[0179] 8.1 Activation and Pre-culture

[0180] Streak the strain cells on a 2% YPD plate and activate them at 30 °C for 24 h. Use an inoculation loop to pick an appropriate amount of cells and inoculate them into a 500 mL conical flask containing 100 mL of 5% YPD. Pre-culture at 30 °C and 160 rpm for 16 h. Centrifuge at 4 °C and 8000×g for 2 min to collect the cells for subsequent fermentation.

[0181] 8.2 Fermentation in Synthetic Medium

[0182] (1) Glucan Medium

[0183] Dispense 10 mL of glucan medium into a 50 mL conical flask. Add the cells displaying cellulase at an initial inoculum of 2 g dry weight / L and perform fermentation in a shaker at 35 °C and 120 rpm. Collect 0.5 mL of the fermentation broth at 0 h, 4 h, 8 h, 12 h, and 24 h for component analysis.

[0184] (2) Xylan Medium

[0185] Dispense 20 mL of xylan medium into a 50 mL conical flask. Add the cells displaying hemicellulase at an initial inoculum of 2 g dry weight / L and perform fermentation in a shaker at 35 °C and 120 rpm. Collect 1 mL of the fermentation broth at 0 h, 4 h, 8 h, 12 h, and 24 h for component analysis.

[0186] (3) Mixed Sugar Medium

[0187] Mix the hemicellulase-displaying strain SEB25 and the cellulase-displaying strain SEB26 in a ratio of 1:1 to obtain composite strain 1 and in a ratio of 7:3 to obtain composite strain 2. Dispense 50 mL of mixed sugar medium into a 250 mL conical flask. Add composite strains 1 and 2 at an initial inoculum of 0.5 g dry weight / L respectively and perform fermentation in a shaker at 35 °C and 120 rpm. Collect 2 mL of the fermentation broth at 0 h, 4 h, 8 h, 12 h, 24 h, and 48 h for component analysis.

[0188] 8.3 Fermentation results

[0189] (1) Fermentation results of dextran medium

[0190] Batch fermentation was carried out using dextran medium to compare the ability of SEB5 (starting strain) and SEB26 (SEB5, co-displaying endo-β-glucanase TrEG2 and exo-β-glucanase TrCBH1) to grow and ferment using dextran. The dextran concentration in the medium inoculated with SEB5 remained almost unchanged, and no glucose was detected. The cells did not grow and did not produce ethanol ( Figure 1 ). In contrast, SEB26 rapidly hydrolyzed dextran (about 6.8 g / L) and released glucose within the first 12 h ( Figure 1 a), producing about 2.4 g / L ethanol ( Figure 1 b). After 12 h, dextran was no longer hydrolyzed, glucose was rapidly consumed, and the cells used ethanol for growth. It can be seen that the cell surface display of cellulase system can endow the strain with the ability to ferment dextran.

[0191] The strain SEB26 obtained in the embodiment of the present invention can simultaneously display endo-β-glucanase and exo-β-glucanase, expanding the substrate spectrum of SEB5, endowing the strain with the ability to metabolize dextran, endowing it with the utilization of oligosaccharides in straw hydrolysate, thus developing a recombinant strain that can directly ferment using oligosaccharides, which is one of the effective means to improve the total sugar utilization rate and reduce the treatment cost of concentrated acid hydrolysis process.

[0192] (2) Fermentation results of xylan medium

[0193] Batch fermentation was carried out using xylan medium to compare the ability of SEB5 (starting strain) and SEB25 (SEB5, co-displaying xylanase AnxlnD and xylosidase AnxlnB) to grow and ferment using xylan.

[0194] A small amount of xylose (less than 0.5 g / L) was detected in the medium inoculated with SEB5. The cells grew slowly and hardly produced ethanol and xylitol ( Figure 2 ). In contrast, SEB25 could hydrolyze xylan into xylose and grow using xylose ( Figure 2 a). After 24 h of fermentation, about 1.66 g / L ethanol and 0.45 g / L xylitol were produced ( Figure 2 b). It can be seen that the cell surface display of hemicellulase system can endow the strain with the ability to metabolize xylan.

[0195] In the embodiments of the present invention, the obtained strain SEB25 can simultaneously display xylanase xlnD and xylosidase xlnB, expanding the substrate spectrum of SEB5, endowing the strain with the ability to metabolize xylan, endowing it with the utilization of oligosaccharides in straw hydrolysate, and thus developing a recombinant strain that can directly utilize oligosaccharides for fermentation, which is one of the effective means to improve the total sugar utilization rate and reduce the treatment cost of the concentrated acid hydrolysis process.

[0196] (3) Fermentation results using concentrated acid straw hydrolysate containing oligosaccharides

[0197] The concentrated acid straw hydrolysate used contained reducing sugars (glucose 60 g / L, xylose 40 g / L), cellobiose (10 g / L), and oligosaccharides (β-glucan 15 g / L, xylan 15 g / L). Batch fermentation was carried out using this hydrolysate to compare the abilities of SEB5, composite strain 1 (SEB25:SEB26 = 1:1), and composite strain 2 (SEB25:SEB26 = 7:3) to grow and ferment using the hydrolysate.

[0198] Results: SEB5 depleted glucose within 8 h of fermentation, almost depleted xylose within 24 h of fermentation, and depleted cellobiose within 48 h of fermentation, producing 40.13 g / L ethanol and 7.43 g / L xylitol ( Figure 3 a and 3b). The composite strains had the same ability to utilize glucose as SEB5, but starting from 8 h of fermentation, the concentrations of glucose, xylose, and cellobiose in the fermentation broth were higher than those of SEB5, probably because the composite strains hydrolyzed glucan and xylan to release cellobiose and monosaccharides. After 48 h of fermentation, composite strain 1 produced 49.7 g / L ethanol and 11.52 g / L xylitol, which were approximately 23.87% and 55.0% higher than those of SEB5, respectively. Composite strain 2 produced 50.34 g / L ethanol and 11.21 g / L xylitol, which were approximately 25.46% higher than those of SEB5, respectively, and the xylitol yield was approximately 50.78% higher. It can be seen that the inoculation ratio of the two strains had no obvious effect on the utilization effect of oligosaccharides.

[0199] It can be known that simultaneously displaying cellulase and hemicellulase on the surface of Saccharomyces cerevisiae cells can effectively utilize the oligosaccharides in straw hydrolysates, thereby improving the total sugar utilization rate. By mixing two yeast strains, a synergistic flora with both cellulase and hemicellulase activities was constructed, which could effectively promote the utilization of oligosaccharides and greatly improve the utilization efficiency of the total sugar in straw hydrolysis.

[0200] Currently, most of the research on improving the utilization of oligosaccharides in straw material hydrolysates focuses on laboratory strains, there are few reports on industrial strains, and there is no research on using composite strains to improve sugar utilization rate. In the limited reports, as attached Figure 4As shown, there have been reports that the industrial brewing yeast ER-X-2P ferments using corncob hot water hydrolysate, with the xylitol yield being 0.112 g / g xylose and the ethanol yield being 0.247 g / g total sugar. Compared with the Figure 4 reports in the appendix, the composite strain constructed in the present invention produces more xylitol and ethanol using oligosaccharides under lower inoculum amounts and shorter fermentation times. Therefore, the composite strain constructed in the present invention has good application potential in the fermentation of cellulosic material hydrolysates.

[0201] The literature sources involved in this patent are as follows:

[0202] [1] Mans R, van Rossum HM, Wijsman M, et al. CRISPR / Cas9: a molecular swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae[J]. FEMS Yeast Research, 2015, 15(2): fov004.

[0203] [2] Zhang GC, Kong II, Kim H, et al. Construction of a quadruple auxotrophic mutant of an industrial polyploid Saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease[J]. Applied and Environmental Microbiology, 2014, 80(24): 7694 - 7701.

[0204] [3] Xie CY, Yang BX, Song QR, et al. Different transcriptional responses of haploid and diploid S. cerevisiae strains to changes in cofactor preference of XR[J]. Microbial Cell Factories, 2020, 19.

[0205] [4]Cunha JT, Romaní A, Inokuma K, et al. Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrial Saccharomyces cerevisiae as efficient whole cell biocatalysts[J]. Biotechnol Biofuels, 2020, 13:138.

[0206] [5]Cunha JT, Gomes DG, Romaní A, et al. Cell surface engineering of Saccharomyces cerevisiae for simultaneous valorization of corn cob and cheese whey via ethanol production[J]. Energy Conversion and Management, 2021, 243:114359。

[0207] The above specific embodiments further elaborate on the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Saccharomyces cerevisiae strain, characterized in that: The brewer's yeast strain is Saccharomyces cerevisiaeSEB26, the preservation number is CGMCC No.33178, the preservation date is December 24, 2024, the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, and the preservation unit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. The method for constructing a Saccharomyces cerevisiae strain according to claim 1, characterized in that: The following steps are involved: Step 1, construct strain SEB5EG: (1) Construction of a surface display gene cassette for β-glucanase EG2; (2) Introducing the Cas9-NAT plasmid into the Saccharomyces cerevisiae strain SEB5 to obtain the strain SEB5-Cas9; (3) The pMEL13-PHO13 plasmid and the β-glucanase EG2 surface display gene cassette were introduced into the strain SEB5-Cas9; (4) removing the Cas9-NAT plasmid and pMEL13-PHO13 plasmid of the correct transformant to obtain strain SEB5 EG; Step 2: Construction of strain SEB26: (5) Construction of pMEL13-ALD6 plasmid and β-glucan exonuclease TrCBH1 surface display gene cassette; (6) Introducing the Cas9-NAT plasmid into the Saccharomyces cerevisiae strain SEB5EG to obtain the strain SEB5EG-Cas9; (7) Using strain SEB5EG-Cas9 as the starting strain, the β-glucan exonuclease TrCBH1 surface display gene cassette and pMEL13-ALD6 plasmid were transferred into cells; (8) The plasmid was removed to obtain strain SEB26.

3. The method for constructing a Saccharomyces cerevisiae strain according to claim 2, characterized in that: The process of constructing the surface display gene cassette of β-glucanase EG2 in (1) is as follows: Synthetic P SED1 -SS SED1 -TrEG2-A SED1 -T SAG1 The gene was ligated to the EcoRI and SalI sites of the pUC57 vector to generate the pUC57-SED1-TrEG2 plasmid. SED1 SS SED1 , TrEG2, A SED1 , T SAG1 The sequences of the genes are SEQ ID NO: 7, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 10, SEQ ID NO: 11; The pUC57-SED1-TrEG2 plasmid was used as a template and primers PHO13celRE-F and PHO13celRE-R were used for amplification. The obtained DNA fragment was the β-glucan endonuclease EG2 surface display gene cassette. The sequences of the primers PHO13celRE-F and PHO13celRE-R were SEQ ID NOs: 16-17.

4. The method for constructing a cerevisiae yeast strain according to claim 2, characterized in that: (3) The obtained strain was verified by colony PCR using primers PHO13-dgF and PHO13-dgR. The sequences of primers PHO13-dgF and PHO13-dgR are SEQ ID NOs: 18-19.

5. The method for constructing a cerevisiae yeast strain according to claim 2, characterized in that: The process of constructing the pMEL13-ALD6 plasmid in (5) is as follows: Construction of double-stranded gRNA fragments: Designing the gRNA recognition sequence within the ALD6 gene coding sequence, synthesizing the primer pair ALD6 TG F / R with a 20 bp recognition sequence and 50 bp upstream and downstream homology arms, and diluting, mixing, heating, and cooling at room temperature the two single-stranded primers in sequence; The linear backbone of the gRNA plasmid was amplified using the pMEL13 plasmid as a template; Connect the double-stranded gRNA fragment and the pMEL13 linear backbone by homologous recombination to obtain the pMEL13-ALD6 plasmid; The sequences of the primer pair ALD6 TG F / R are SEQ ID NOs: 1-2; In the process of amplifying the linear backbone of the gRNA plasmid, the primer sequences of the plasmid backbone are shown in SEQ ID NOs: 3 to 4.

6. The method for constructing a cerevisiae yeast strain according to claim 2, characterized in that: The process of constructing the surface display gene cassette of β-glucan exonuclease TrCBH1 in (5) is as follows: Synthetic P SED1 -SS SED1 -TrCBH1-A SED1 -T SAG1 gene and connected it to the EcoRI and SalI sites of the pUC57 vector to obtain the pUC57-SED1-TrCBH1 plasmid, and the pUC57-SED1-TrCBH1 plasmid was used as a template and amplified using primers ALD6celRE-F and ALD6 celRE-R. The obtained DNA fragment was the β-glucan exonuclease TrCBH1 surface display gene cassette; The sequences of the TrCBH1 genes are SEQ ID NO: 22; The sequences of primers ALD6 celRE-F and ALD6 celRE-R are SEQ ID NOs: 5 to 6, respectively.

7. The method for constructing a cerevisiae yeast strain according to claim 2, characterized in that: (7) The obtained strain was verified by colony PCR using primers ALD6-dgF and ALD6-dgR. The sequences of primers ALD6-dgF and ALD6-dgR are SEQ ID NOs: 12-13.

8. Use of the Saccharomyces cerevisiae strain SEB26 obtained according to the method of claim 1 or any one of claims 2 to 7 in the production of ethanol by fermentation using oligosaccharides.

9. A composite strain, characterized in that: The method comprises the Saccharomyces cerevisiae SEB26 strain as claimed in claim 1 or the Saccharomyces cerevisiae SEB26 strain constructed according to any one of claims 2 to 7, and further comprises the Saccharomyces cerevisiae SEB25 strain, wherein the deposit number of the Saccharomyces cerevisiae SEB25 strain is CGMCC No.33177.

10. A composite strain according to claim 9, characterized in that: The mixing ratio of SEB25 strain and SEB26 strain is 1:1 to 7:3.

Images