Saccharomyces cerevisiae strain SEB25, construction method, synthetic flora and application

Abstract

The invention discloses a saccharomyces cerevisiae strain SEB25, a construction method, a synthetic flora and application, the preservation number of the saccharomyces cerevisiae strain SEB25 is CGMCC (China General Microbiological Culture Collection Center) No.33177, the preservation date is December 24, 2024, and the preservation unit is China General Microbiological Culture Collection Center. The SEB25 strain can display xylanase xlnD and xylosidase xlnB at the same time, the strain is endowed with the xylan metabolism capacity and the utilization capacity for oligosaccharide in straw hydrolysate, and a recombinant strain capable of directly utilizing oligosaccharide for fermentation is developed. The synthetic flora provided by the invention comprises SEB25 and SEB26 strains which respectively express specific hemicellulase (xylanase and xylosidase) and cellulase (glucosan endonuclease and glucosan exonuclease), straw concentrated acid hydrolysate containing oligosaccharide is used for fermentation, and the yield of ethanol can be remarkably increased.

Description

Technical Field

[0001] The present invention relates to the technical field of Saccharomyces cerevisiae strains, and specifically relates to the Saccharomyces cerevisiae strain SEB25, its construction method, a synthetic microbial community containing the Saccharomyces cerevisiae strain SEB25, and the application of this synthetic microbial community. 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. As a traditional ethanol-producing strain, Saccharomyces cerevisiae has become an ideal choice for cellulose fuel ethanol production due to its excellent biosafety, strong stress resistance, and easy gene manipulability.

[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, pretreatment is usually required to destroy its complex structure 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 ferment ethanol using glucose, xylose, and cellobiose. However, this strain cannot ferment oligosaccharides, 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 SEB25, its construction method, a synthetic microbial community containing the Saccharomyces cerevisiae strain SEB25, and the application of this synthetic microbial community.

[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 SEB25, which is Saccharomyces cerevisiae SEB25, with a preservation number of CGMCC No. 33177, a preservation date of December 24, 2024, and a preservation unit of the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the address of the preservation unit being 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 SEB25, which includes the following steps:

[0010] Step 1, constructing strain SEB5AD:

[0011] (1) Constructing the pMEL13-ALD6 plasmid and the xylanase xlnD surface display gene cassette;

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

[0013] (3) Introducing the pMEL13-ALD6 plasmid and the xylanase xlnD surface display gene cassette into strain SEB5-Cas9;

[0014] (4) Removing the Cas9-NAT plasmid and the pMEL13-ALD6 plasmid from the correct transformant to obtain strain SEB5AD;

[0015] Step 2, constructing strain SEB25:

[0016] (5) Constructing the pMEL13-PHO13 plasmid and the xylosidase xlnB surface display gene cassette;

[0017] (6) Introducing the Cas9-NAT plasmid into the Saccharomyces cerevisiae strain SEB5AD to obtain strain SEB5AD-Cas9;

[0018] (7) Using strain SEB5AD-Cas9 as the starting strain, transferring the xylosidase xlnB surface display gene cassette and the pMEL13-PHO13 plasmid into the cells;

[0019] (8) Removing the plasmid to obtain strain SEB25.

[0020] As a preferred design, the process of constructing the pMEL13-ALD6 plasmid in (1) is:

[0021] Constructing double-stranded gRNA fragments: Design gRNA recognition sequences within the ALD6 gene coding sequence, synthesize primer pair ALD6 TG F / R with a 20-bp recognition sequence and 50-bp homologous arms upstream and downstream, dilute, mix, heat, and cool the two single-stranded primers successively at room temperature;

[0022] Amplify the linear backbone of the gRNA plasmid using the pMEL13 plasmid as a template;

[0023] Connect the double-stranded gRNA fragment and the pMEL13 linear backbone by homologous recombination to obtain the pMEL13-ALD6 plasmid;

[0024] The sequences of primer pair ALD6 TG F / R are SEQ ID NO: 1-2;

[0025] 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.

[0026] As a preferred design, the process of constructing the xylanase xlnD surface display gene cassette in (1) is as follows:

[0027] Synthesize P SED1 -S.S AnxlnD -AnxlnD-A SED1 -T SAG1 gene and ligate it to the EcoRI and SalI sites of the pUC57 vector to obtain the pUC57-SED1-AnxlnD plasmid. The sequences of P SED1 、S.S AnxlnD 、AnxlnD、A SED1 、T SAG1 genes are SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 respectively;

[0028] Using the pUC57-SED1-AnxlnD plasmid as a template, amplify using primers ALD6celRE-F and ALD6celRE-R. The obtained DNA fragment is the xylanase xlnD surface display gene cassette. The sequences of primers ALD6celRE-F and ALD6celRE-R are SEQ ID NO: 5-6.

[0029] As a preferred design, in (3), the obtained strain is verified by colony PCR. The verification is carried out using primers ALD6-dgF and ALD6-dgR. The sequences of primers ALD6-dgF and ALD6-dgR are SEQ ID NO: 12-13.

[0030] As a preferred design, the process of constructing the xylosidase xlnB surface display gene cassette in (5) is as follows:

[0031] Synthesize P SED1 -S.S AnxlnB -AnxlnB-A SED1 -T SAG1 gene and ligate it to the EcoRI and SalI sites of the pUC57 vector to obtain the pUC57-SED1-AnxlnB plasmid. Using the pUC57-SED1-AnxlnB plasmid as a template, amplify it with primers PHO13celRE-F and PHO13celRE-R. The obtained DNA fragment is the xylosidase xlnB surface display gene cassette;

[0032] S.S AnxlnB The sequences of S.S and AnxlnB genes are SEQ ID NO: 14 and SEQ ID NO: 15 respectively;

[0033] The sequences of primers PHO13celRE-F and PHO13celRE-R are SEQ ID NO: 16-17 respectively.

[0034] As a preferred design, the obtained strain is verified by colony PCR in (7). The verification is carried out using primers PHO13-dgF and PHO13-dgR, and the sequences of primers PHO13-dgF and PHO13-dgR are SEQ ID NO: 18-19.

[0035] The third object of the present invention is to provide a synthetic flora, including the Saccharomyces cerevisiae SEB25 strain as described above or the Saccharomyces cerevisiae SEB25 strain constructed by any of the above methods, and also including the Saccharomyces cerevisiae SEB26 strain. The preservation number of the Saccharomyces cerevisiae SEB26 strain is CGMCC No. 33178.

[0036] As a preferred design, the mixing ratio of the SEB25 strain to the SEB26 strain is 1:1-7:3.

[0037] The fourth object of the present invention is to provide the application of the above-mentioned synthetic flora in straw concentrated acid hydrolysis liquor. The straw concentrated acid hydrolysis liquor used contains oligosaccharides, and also contains reducing sugars and cellobiose;

[0038] Preferably, the concentration of oligosaccharides in the straw concentrated acid hydrolysis liquor is 15 g / L of β-glucan and 15 g / L of xylan.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0040] 1. The strain SEB25 provided by the embodiment of the present invention can simultaneously display xylanase xlnD and xylosidase xlnB, expanding the substrate spectrum of SEB5, endowing the strain with the ability to metabolize xylan, and endowing it with the ability to utilize oligosaccharides in straw hydrolysis liquor. Thus, a recombinant strain that can directly utilize oligosaccharides for fermentation is developed, 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.

[0041] 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 (β-glucanase and exo-β-glucanase) are respectively expressed on the cell surface of the SEB5 strain, and these two recombinant strains are combined and fermented with the concentrated acid hydrolysis liquor of straw containing oligosaccharides. The results show that this combination strategy can significantly improve the ethanol yield. The strains constructed in this study can also be applied to the ethanol fermentation in the process of "pretreatment + enzymatic hydrolysis", reducing the input of cellulase. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] 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 thus 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:

[0043] Figure 1 For the xylan fermentation results in Example 1, a represents the dry cell weight (DCW) and xylose concentration (Xylose); b represents the ethanol and xylitol concentrations.

[0044] Figure 2 For the glucan fermentation results in Example 2, a represents the glucan (Glucan) and glucose (Glucose) concentrations; b represents the dry cell weight and ethanol concentration.

[0045] Figure 3 For the fermentation results of the concentrated acid hydrolysis liquor of straw in Example 2, a represents the glucose, xylose and cellobiose (Cellobiose) concentrations; b represents the dry cell weight, ethanol and xylitol concentrations.

[0046] Figure 4 For the comparison chart of the fermentation performance of the cell surface-displaying cellulase strains between the disclosed technology and the present patent application. Detailed implementation mode

[0047] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings. The illustrative implementation modes of the present invention and their descriptions are only used to explain the present invention and do not limit the present invention.

[0048] Embodiment 1:

[0049] The preservation information of the Saccharomyces cerevisiae strain SEB25 is as follows:

[0050] The preservation number is CGMCC No. 33177, the preservation date is December 24, 2024, the preservation unit is the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the address of the preservation unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0051] SEB25 was constructed by the following method:

[0052] 1. The starting strain for the construction process is SEB5, which is preserved in the General Microbiology Center of the China Microbial Culture Collection Management Committee, with the preservation number CGMCC11325.

[0053] 2. Culture medium

[0054] 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 cools to 50 - 60 °C, antibiotics are added.

[0055] Table 1 Composition of the culture media used

[0065]

[0066]

[0067] 3. Plasmids, strains and primers

[0068] 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.

[0069] Table 2 Plasmid and strain information

[0070]

[0071] Note: P: Promoter; S.S: Signal Sequence; A: Anchor; T: Terminator.

[0072] Table 3 Primers for constructing gRNA plasmids

[0073]

[0074] Note: F: Forward primer; R: Reverse primer; Underline: 20bp recognition sequence.

[0075] Table 4 Primers required for strain transformation

[0076]

[0077]

[0078] Note: RE: Repair fragment; dg: Verification primer; F: Forward primer; R: Reverse primer

[0079] 4. Construction of strain SEB5AD

[0080] 4.1 Construction of gRNA plasmid pMEL13-ALD6

[0081] 1) Construction of double-stranded gRNA fragment

[0082] Using the genomic information of the model yeast S288c as a reference, a gRNA recognition sequence (20bp) was designed within the coding sequence of the ALD6 gene using the CHOPCHOP website. A primer pair ALD6 TG F / R (sequences are shown in Table 3) with a 20bp recognition sequence and 50bp homologous arms at both the upstream and downstream was synthesized. The two single-stranded primers were separately diluted to 10μM with sterile water, mixed in equal volumes, heated at 95°C for 5 min, and cooled to room temperature to obtain a double-stranded gRNA fragment.

[0083] 2) Amplification of the linear backbone of the gRNA plasmid

[0084] Using the pMEL13 plasmid as a template, the linear backbone was amplified. The PCR reaction system and reaction conditions are shown in Table 5 below. After purification of the PCR product, the linear backbone of the gRNA plasmid was obtained.

[0085] Table 5 PCR amplification of the gRNA linear backbone

[0086]

[0087]

[0088] Digest the template plasmid 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 amount of plasmid (1 μg plasmid plus 1 μL DpnI). Purify to obtain the gRNA linear backbone.

[0089] Table 6 gRNA Linear Backbone Template Digestion System

[0090]

[0091] 3) Gibson Ligate the gRNA fragment and the linear backbone

[0092] Ligate the double-stranded gRNA fragment obtained in 1) and the pMEL13 linear backbone obtained in 2) 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. Spread the bacterial solution on an LB + Kan plate and culture it overnight at 37 °C. Inoculate the transformant into a test tube containing 5 mL of LB + Kan liquid medium and culture it for 12 - 16 h (160 rpm, 37 °C). Collect the bacterial cells and extract the gRNA plasmid. Sequence to confirm the correct gRNA plasmid pMEL13-ALD6 is obtained.

[0093] Table 7 Gibson Ligation Reaction System

[0094]

[0095] 4.2 Construction of the Repair Fragment - Xylanase xlnD Surface Display Gene Cassette

[0096] Entrust a company to synthesize the P SED1 -S.S AnxlnD -AnxlnD-A SED1 -T SAG1 gene and ligate it 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 (synthesized by an entrusted gene synthesis company), 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, use primers ALD6celRE-F and ALD6celRE-R (primer sequences are shown in Table 4 above) for amplification. 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.

[0097] Table 8 Xylanase xlnD Surface Display Gene Cassette Sequence

[0098]

[0099]

[0100]

[0101]

[0102] Table 9 PCR Amplification and Repair Fragments

[0103]

[0104] 4.3 Preparation of Cas9-NAT Plasmid

[0105] 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 bacterial cells and extract the Cas9-NAT plasmid.

[0106] 4.4 CRISPR / Cas9 Transformation

[0107] (1) Introduce the Cas9-NAT plasmid into the Saccharomyces cerevisiae strain by the lithium acetate transformation method

[0108] a. Streak the strain SEB5 on a 2% YPD plate for activation. Take an appropriate amount of bacterial cells and inoculate them into 5 mL of 2% YPD liquid medium, and culture them with shaking at 30 °C and 160 rpm for 16 h;

[0109] b. Take 2 mL of the bacterial liquid and inoculate it into 100 mL of 2% YPD culture medium, and culture it at 30 °C and 160 rpm for 2 - 3 h. When the OD 600 reaches 0.2 - 0.3, centrifuge to collect all the bacterial cells. Wash the bacterial cell pellet twice with sterile water and then resuspend it in 300 μL of sterile water, and place it on ice for later use;

[0110] c. Boil salmon sperm DNA (ssDNA) at 100 °C for 5 min and place it on ice for later use;

[0111] d. 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) to a 1.5 mL centrifuge tube in sequence. After mixing, perform heat shock at 42 °C for 40 min;

[0112] 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;

[0113] f. Centrifuge at 8,000×g for 1 min, discard the supernatant, and resuspend the cells in 1 mL of sterile water. Take 100 μL of the bacterial solution and spread it on a 2% YPD + NAT plate, and culture it at 30 °C for 2 - 3 d;

[0114] g. Streak the transformants on a 2% YPD + NAT plate. The transformants that can grow contain the Cas9 - NAT plasmid.

[0115] (2) Knock - in the xylanase xlnD surface display gene cassette at the ALD6 locus

[0116] a. Streak the strain SEB5 - Cas9 containing the Cas9 - NAT plasmid on a 2% YPD + NAT plate for activation. Take an appropriate amount of the cells and inoculate them into 5 mL of 2% YPD + NAT liquid medium, and culture them at 30 °C with shaking at 160 rpm for 16 h;

[0117] b. Take 2 mL of the bacterial solution and inoculate it into 100 mL of 2% YPD + NAT culture medium, and culture it at 30 °C with shaking at 160 rpm for 2 - 3 h. When the OD 600 reaches 0.2 - 0.3, centrifuge to collect all the cells. Wash the cell pellet twice with sterile water and then resuspend it in 300 μL of sterile water, and place it on ice for later use;

[0118] c. Boil salmon sperm DNA (ssDNA) at 100 °C for 5 min, and place it on ice for later use;

[0119] d. In a 1.5 mL centrifuge tube, sequentially add 50% PEG4000 (240 μL), 4 M lithium acetate solution (9 μL), ssDNA (25 μL), pMEL13 - ALD6 plasmid (600 ng), xlnD repair fragment (1.5 - 2.5 μg), add sterile water to make up to 351 μL, add yeast cells (50 μL), mix well and then heat - shock at 42 °C for 40 min;

[0120] 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 it at 30 °C with shaking at 160 rpm for 2 - 3 h;

[0121] f. Centrifuge at 8,000×g for 1 min, discard the supernatant, resuspend the cells in 150 μL of sterile water, spread all the bacterial solution on a 2% YPD + NAT + G418 plate, culture it at 30 °C for 2 - 3 d, and pick the transformants for colony PCR verification.

[0122] 4.5 Colony PCR verification

[0123] a. In a 1.5 mL centrifuge tube, sequentially add 95 μL of 1% SDS, 5 μL of 4 M lithium acetate solution and an appropriate amount of cells, vortex and then incubate at 75 °C for 10 min;

[0124] b. Add 300 μL of absolute ethanol to the centrifuge tube and 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;

[0125] c. Add 100 μL of sterile water, vortex, centrifuge at 13,000 rpm at room temperature for 1 min, and retain the supernatant;

[0126] 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;

[0127] 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.

[0128] Table 10 Colony PCR verification system

[0129]

[0130] 4.6 Plasmid removal

[0131] Remove the Cas9-NAT plasmid and pMEL13-ALD6 plasmid of the correct transformant. The specific steps are as follows:

[0132] a. Streak the correct transformant 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.

[0133] 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;

[0134] 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; the strain that can only grow on the 2% YPD plate has successfully removed the plasmid. Finally, the strain SEB5AD that successfully displayed xylanase on the cell surface was obtained.

[0135] 5 Construction of strain SEB25

[0136] 5.1 Construction of the repair fragment - xylosidase xlnB surface display gene cassette

[0137] Entrust the company to synthesize P SED1 -S.S AnxlnB -AnxlnB-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-AnxlnB. Among them, AnxlnB was derived from Aspergillus niger and codon-optimized during synthesis. The base sequence is shown in Table 11 below. Using the pUC57-SED1-AnxlnB plasmid as a template, amplification was performed using primers PHO13celRE-F and PHO13celRE-R (primer sequences are shown in Table 4), and the obtained DNA fragment is the xylanase 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.

[0138] Table 11 Xylanase xlnB surface display gene cassette sequence

[0139]

[0140] Note: The sequences of other components are the same as in Table 8.

[0141] 5.2 Preparation of pMEL13-PHO13 plasmid

[0142] E. coli containing the pMEL13-PHO13 plasmid was inoculated into 5 mL of LB+KAN liquid medium and cultured with shaking at 37 °C and 160 rpm for 12 - 16 h. The cells were collected by centrifugation, and the pMEL13-PHO13 plasmid was extracted.

[0143] 5.3 CRISPR / Cas9 transformation

[0144] (1) The Cas9-NAT plasmid was introduced into the Saccharomyces cerevisiae strain by the lithium acetate transformation method

[0145] Using SEB5AD as the starting strain, the lithium acetate transformation process was the same as in 4.4(1), and the SEB5AD-Cas9 strain was obtained.

[0146] (2) Knock in the xylanase xlnB surface display gene cassette at the PHO13 locus

[0147] Using SEB5AD-Cas9 as the starting strain, the xlnB surface display gene cassette P SED1 -S.S AnxlnB -AnxlnB-A SED1 -T SAG1 and the pMEL13-PHO13 plasmid were transferred into the cells. The detailed steps were the same as in 4.4(2).

[0148] 5.4 Colony PCR verification

[0149] 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 PHO13-dgF and PHO13-dgR. The reaction system and reaction conditions are shown in Table 10.

[0150] 5.5 Plasmid removal

[0151] The operation steps are the same as those in 4.6. Finally, strain SEB25 that simultaneously displays xylanase xlnD and xylosidase xlnB on the cell surface is successfully obtained.

[0152] 6. Fermentation evaluation

[0153] 6.1 Activation and pre-culture

[0154] Streak the strain cells on a 2% YPD plate and activate them at 30 °C for 24 h. Use an inoculation loop to pick up an appropriate amount of cells and inoculate them into a 500 mL conical flask containing 100 mL of 5% YPD, and pre-culture them 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.

[0155] 6.2 Fermentation in synthetic medium

[0156] Xylan medium: Aliquot 20 mL of xylan medium into a 50 mL conical flask, and add cells displaying hemicellulase on the surface at an initial inoculum of 2 g dry weight / L, and ferment in a shaker at 35 °C and 120 rpm. Collect 1 mL of fermentation broth at 0 h, 4 h, 8 h, 12 h, and 24 h for component analysis.

[0157] 6.3 Fermentation results in xylan medium

[0158] Perform batch fermentation using xylan medium to compare the abilities of SEB5 (starting strain) and SEB25 (SEB5, co-displaying xylanase AnxlnD and xylosidase AnxlnB) to grow and ferment using xylan.

[0159] A small amount of xylose (less than 0.5 g / L) was detected in the medium inoculated with SEB5, the cell growth was slow, and almost no ethanol and xylitol were produced ( Figure 1 ). In contrast, SEB25 could hydrolyze xylan into xylose and grow using xylose ( Figure 1 a). After 24 h of fermentation, approximately 1.66 g / L of ethanol and 0.45 g / L of xylitol were produced ( Figure 1 b). Thus, it can be seen that displaying the hemicellulase system on the cell surface can endow the strain with the ability to metabolize xylan.

[0160] The strain SEB25 obtained in this example 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 ability to utilize oligosaccharides in straw hydrolysate, 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.

[0161] Example 2:

[0162] The present invention also provides a synthetic consortium, which includes the Saccharomyces cerevisiae SEB25 strain as in Example 1, and also includes the Saccharomyces cerevisiae SEB26 strain. The preservation number of the SEB26 strain is CGMCC No. 33178, the preservation date is December 24, 2024, the preservation unit is the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the address of the preservation unit is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0163] The SEB26 strain can display cellulases (endo-β-glucanase and exo-β-glucanase), can effectively utilize glucan, and in combination with the SEB25 strain, can ferment the straw concentrated acid hydrolysate containing oligosaccharides. This combination strategy can significantly increase the ethanol yield.

[0164] The SEB26 strain was constructed according to the following method:

[0165] Construction of strain SEB5EG

[0166] 7.1 Construction of the repair fragment - the surface display gene cassette of endo-β-glucanase EG2

[0167] Entrust a company to synthesize the P SED1 -S.S SED1 -TrEG2-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-TrEG2. Among them, TrEG2 is derived from Trichoderma reesei (synthesized by a gene synthesis company), and codon optimization was carried out during synthesis. The base sequence is shown in Table 12 below. Using the pUC57-SED1-TrEG2 plasmid as a template, amplify using primers PHO13celRE-F and PHO13celRE-R (the primer sequences are shown in Table 4). The obtained DNA fragment is the surface display gene cassette of endo-β-glucanase EG2 (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.

[0168] Table 12 β-glucan endonuclease EG2 surface display gene cassette sequence

[0169]

[0170]

[0171] Note: The sequences of other components are the same as those in Table 8.

[0172] 7.2 Knock-in of the β-glucan endonuclease EG2 surface display gene cassette at the PHO13 locus

[0173] Using the strain SEB5-Cas9 containing the Cas9-NAT plasmid as the starting strain, transfer the EG2 surface display gene cassette P SED1 -S.S SED1 -TrEG2-A SED1 -T SAG1 and the pMEL13-PHO13 plasmid into the cells. The detailed steps are the same as in 4.4(2).

[0174] 7.3 Colony PCR verification

[0175] The steps for extracting genomic DNA from the cells 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.

[0176] 7.4 Plasmid removal

[0177] The operation steps are the same as in 4.6. Finally, successfully obtain the strain SEB5EG that displays β-glucan endonuclease EG2 on the cell surface.

[0178] 8 Construction of strain SEB26

[0179] 8.1 Construction of the repair fragment - β-glucan exohydrolase TrCBH1 surface display gene cassette

[0180] Entrust the company to synthesize P SED1 -S.S SED1 -TrCBH1-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-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 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.

[0181] Table 13 Sequence of the exoglucanase TrCBH1 surface display gene cassette

[0182]

[0183]

[0184] Note: The sequences of other components are the same as those in Table 8.

[0185] 8.2 CRISPR / Cas9 transformation

[0186] (1) The Cas9-NAT plasmid was introduced into the Saccharomyces cerevisiae strain by the lithium acetate transformation method

[0187] Using SEB5EG as the starting strain, the lithium acetate transformation process is shown in 4.4(1), and the SEB5EG-Cas9 strain was obtained.

[0188] (2) Knock in the exoglucanase TrCBH1 surface display gene cassette at the ALD6 locus

[0189] 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 were transferred into the cells. The detailed steps are the same as 4.4(2).

[0190] 8.3 Colony PCR verification

[0191] The steps for extracting the genomic DNA of the cells are the same as 4.5. Take 1 μL as the template, and PCR verification was performed using primers ALD6-dgF and ALD6-dgR. The reaction system and reaction conditions are shown in Table 10.

[0192] 8.4 Removal of the plasmid

[0193] The operation steps are the same as in 4.6, and finally the strain SEB26 that simultaneously displays endoglucanase EG2 and exoglucanase TrCBH1 on the cell surface is successfully obtained.

[0194] 9 Fermentation evaluation

[0195] 9.1 Activation and preculture

[0196] Streak the strain cells on a 2% YPD plate and activate them at 30 °C for 24 h. Use an inoculation loop to pick up an appropriate amount of cells and inoculate them into a 500 mL conical flask containing 100 mL of 5% YPD, and preculture them 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.

[0197] 9.2 Fermentation in synthetic medium

[0198] (1) Glucan medium

[0199] 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 ferment in a shaker at 35 °C and 120 rpm. Collect 0.5 mL of fermentation broth at 0 h, 4 h, 8 h, 12 h, and 24 h for component analysis.

[0200] (2) Mixed sugar medium

[0201] Mix the hemicellulase-displaying strain SEB25 and the cellulase-displaying strain SEB26 in Example 1 in a ratio of 1:1 to obtain synthetic microbial community 1, and in a ratio of 7:3 to obtain synthetic microbial community 2. Dispense 50 mL of mixed sugar medium into a 250 mL conical flask, add synthetic microbial communities 1 and 2 at an initial inoculum of 0.5 g dry weight / L respectively, and ferment in a shaker at 35 °C and 120 rpm. Collect 2 mL of fermentation broth at 0 h, 4 h, 8 h, 12 h, 24 h, and 48 h for component analysis.

[0202] 9.3 Fermentation results in glucan medium

[0203] Perform batch fermentation using the glucan medium to compare the ability of SEB5 (the starting strain) and SEB26 (SEB5, co-displaying endoglucanase TrEG2 and exoglucanase TrCBH1) to grow and ferment using glucan. The glucan 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 2)。In contrast, SEB25 rapidly hydrolyzes dextran (about 6.8 g / L) and releases glucose within the first 12 h, producing about 2.4 g / L ethanol. After 12 h, dextran is no longer hydrolyzed, glucose is rapidly consumed, and the cells use 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.

[0204] 9.4 Fermentation results using concentrated acid hydrolysis liquor of straw containing oligosaccharides

[0205] The concentrated acid straw hydrolysis liquor used contains reducing sugars (glucose 60 g / L, xylose 40 g / L), cellobiose (10 g / L) and oligosaccharides (β-dextran 15 g / L, xylan 15 g / L). Batch fermentation was carried out using this hydrolysis liquor to compare the abilities of SEB5, synthetic community 1 (SEB25:SEB26 = 1:1) and synthetic community 2 (SEB25:SEB26 = 7:3) to grow and ferment using the hydrolysis liquor.

[0206] 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 ). The synthetic community 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 synthetic community hydrolyzed dextran and xylan to release cellobiose and monosaccharides. After 48 h of fermentation, synthetic community 1 produced 49.7 g / L ethanol and 11.52 g / L xylitol, which were about 23.87% and 55.0% higher than SEB5 respectively. Synthetic community 2 produced 50.34 g / L ethanol and 11.21 g / L xylitol, which were about 25.46% higher than SEB5 respectively, and the xylitol yield was about 50.78% higher. It can be seen that the inoculation ratio of the two strains has no obvious effect on the utilization effect of oligosaccharides.

[0207] It can be known that simultaneously displaying cellulase and hemicellulase on the surface of Saccharomyces cerevisiae cells can effectively utilize the oligosaccharides in the straw hydrolysis products, thereby improving the total sugar utilization rate. By mixing two yeast strains, a synergistic community 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.

[0208] At present, most of the studies on improving the utilization of oligosaccharides in straw material hydrolysis liquor focus on laboratory strains, there are few reports on industrial strains, and there are no studies on improving sugar utilization rate using synthetic communities. In the limited reports, such as attached Figure 4As shown, there are currently reported that the industrial brewing yeast ER-X-2P ferments using the hot water hydrolysate of corncobs, 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 reported in the appendix, the synthetic microbial community constructed in the present invention produces more xylitol and ethanol from oligosaccharides under lower inoculum amounts and shorter fermentation times. Therefore, the synthetic microbial community constructed in the present invention has good application potential in the fermentation of cellulose material hydrolysates.

[0209] The literature sources involved in this patent are as follows:

[0210] [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.

[0211] [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.

[0212] [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.

[0213] [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.

[0214] [5] Cunha JT, Gomes DG, Roman 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.

[0215] The above specific embodiments have further elaborated on the purpose, technical solutions, 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 principles of the present invention shall 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 cerevisiaeSEB25, the preservation number is CGMCC No.33177, the preservation date is December 24, 2024, the preservation unit is the General Microbiology Center of the China 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 SEB5AD: (1) Construction of pMEL13-ALD6 plasmid and xylanase xlnD surface display gene cassette; (2) Introducing the Cas9-NAT plasmid into the Saccharomyces cerevisiae strain SEB5 to obtain the strain SEB5-Cas9; (3) The pMEL13-ALD6 plasmid and the xylanase xlnD surface display gene cassette were introduced into the strain SEB5-Cas9; (4) removing the Cas9-NAT plasmid and pMEL13-ALD6 plasmid of the correct transformant to obtain strain SEB5AD; Step 2: Construction of strain SEB25: (5) Construction of pMEL13-PHO13 plasmid and xylosidase xlnB surface display gene cassette; (6) Introducing the Cas9-NAT plasmid into the Saccharomyces cerevisiae strain SEB5AD to obtain the strain SEB5AD-Cas9; (7) Using strain SEB5AD-Cas9 as the starting strain, the xylosidase xlnB surface display gene cassette and pMEL13-PHO13 plasmid were transferred into cells; (8) The plasmid is removed to obtain strain SEB25.

3. The method for constructing a saccharomyces cerevisiae strain according to claim 2, characterized in that: The process of constructing the pMEL13-ALD6 plasmid in (1) 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.

4. The method for constructing a cerevisiae yeast strain according to claim 2, characterized in that: The process of constructing the xylanase xlnD surface display gene cassette in (1) is as follows: Synthetic P SED1 -SS AnxlnD -AnxlnD-A SED1 -T SAG1 The gene was ligated to the EcoRI and SalI sites of the pUC57 vector to generate the pUC57-SED1-AnxlnD plasmid. SED1 SS AnxlnD 、AnxlnD、A SED1 、T SAG1 The sequences of the genes are SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; The pUC57-SED1-AnxlnD plasmid was used as a template and primers ALD6celRE-F and ALD6celRE-R were used for amplification. The obtained DNA fragment was the xylanase xlnD surface display gene cassette. The sequences of the primers ALD6celRE-F and ALD6celRE-R were SEQ ID NOs: 5-6.

5. 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 ALD6-dgF and ALD6-dgR. The sequences of primers ALD6-dgF and ALD6-dgR are SEQ ID NOs: 12-13.

6. The method for constructing a cerevisiae yeast strain according to claim 2, characterized in that: The process of constructing the xylosidase xlnB surface display gene cassette in (5) is as follows: Synthetic P SED1 -SS AnxlnB -AnxlnB-A SED1 -T SAG1 gene and connected it to the EcoRI and SalI sites of the pUC57 vector to obtain the pUC57-SED1-AnxlnB plasmid, and the pUC57-SED1-AnxlnB plasmid was used as a template and amplified using primers PHO13celRE-F and PHO13celRE-R. The obtained DNA fragment was the xylosidase xlnB surface display gene cassette; SS AnxlnB The sequences of the , AnxlnB genes are SEQ ID NO: 14, SEQ ID NO: 15, respectively; The sequences of primers PHO13celRE-F and PHO13celRE-R are SEQ ID NOs: 16 to 17, 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 PHO13-dgF and PHO13-dgR. The sequences of primers PHO13-dgF and PHO13-dgR are SEQ ID NOs: 18-19.

8. A synthetic bacterial community, characterized in that The method comprises the Saccharomyces cerevisiae SEB25 strain as claimed in claim 1 or the Saccharomyces cerevisiae SEB25 strain constructed according to any one of claims 2 to 7, and further comprises the Saccharomyces cerevisiae SEB26 strain, wherein the deposit number of the Saccharomyces cerevisiae SEB26 strain is CGMCC No.33178.

9. A synthetic bacterial community according to claim 8, characterized in that: The mixing ratio of SEB25 strain and SEB26 strain is 1:1 to 7:

3.

10. Use of a synthetic bacterial consortium as claimed in claim 8 in concentrated acid hydrolyzate of straw, wherein the concentrated acid hydrolyzate of straw contains oligosaccharides, reducing sugars and cellobiose; Preferably, the concentration of oligosaccharides in the concentrated acid hydrolyzate of straw is 15 g / L of β-glucan and 15 g / L of xylan.

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