Application of editing the ptoESK1 gene to improve sugar production efficiency and / or ethanol yield in poplar

Abstract

This invention discloses the application of editing the ptoESK1 gene in improving sugar production efficiency and / or ethanol yield in poplar trees. The invention utilizes CRISPR / Cas9 technology to edit PtoESK1 in Populus tomentosa, resulting in transgenic plants with significantly improved cell wall enzymatic conversion efficiency for sugar production and ethanol yield. This invention lays the foundation for genetic improvement of plant cell walls and targeted molecular breeding, provides new gene resources for plant genetic engineering, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of editing the ptoESK1 gene in improving sugar production efficiency and / or ethanol yield in poplar trees. Background Technology

[0002] Energy shortages and environmental pollution are among the major problems facing the world today. Bioenergy, as a green, sustainable, and clean energy source, offers hope and direction for solving these problems. Using lignocellulose as a raw material, converting it into biofuels such as ethanol through processes like hydrolysis and fermentation is currently the main development direction of bioenergy. Wood provides a large amount of raw materials for chemical and bioenergy production, possessing the advantages of being renewable, abundant, and inexpensive. Wood is mainly composed of plant cell walls, containing cellulose, hemicellulose, and lignin. In bioenergy processes, brewer's yeast mainly utilizes hexoses as a carbon source (Hahn-Hägerdal et al 2007), therefore, the yield of hexoses during biomass pretreatment enzymatic hydrolysis is crucial in the entire degradation pathway. Since the main source of hexoses in plant cell walls is cellulose, the enzymatic hydrolysis efficiency of cellulose directly determines the yield of bioethanol (Van Maris et al 2006). However, plant cell walls are complex network structures, with various macromolecular polymers interacting and cross-linking to form intricate network structures. In the development and utilization of wood, a series of biological, physical, or chemical pretreatment methods are needed to break the cross-links between cell wall molecules. However, this series of pretreatment processes brings huge energy inputs, resulting in very low net energy output efficiency. Furthermore, the pretreatment process is prone to generating secondary pollutants such as waste acid and waste alkali, which seriously hinders the industrial utilization of bioenergy.

[0003] Studies have shown that hemicellulose acts as a bridge between lignin and cellulose components. Hemicellulose covers the surface of cellulose, forming hydrogen bonds with cellulose and covalent bonds with lignin, creating a physical barrier that affects the accessibility of cellulose by cellulase and hinders its degradation (Palonen et al. 2004; Himmel et al. 2007). This cross-linking is closely related to the acetylation modification of hemicellulose. Analysis of hemicellulose from hardwoods (birch) and softwoods (spruce) revealed different acetyl substitution patterns. When acetylation occurs on the main chain residues of xylan in a 2-fold helical conformation, xylan can be tightly anchored to the hydrophilic side of cellulose, indicating that hemicellulose acetylation is crucial for cross-linking between cell wall components. Furthermore, the extensive acetylation modification of hemicellulose not only tightly binds cell wall components, spatially hindering the action of cellulase on cellulose, but also leads to the conversion of acetyl groups into large amounts of acetic acid during pretreatment. This acetic acid is toxic to yeast, inhibiting the sugar alcohol conversion process during fermentation, and is also corrosive to mechanical equipment (Chen et al 2012; Xiong et al 2013; deCarvalho et al 2015). Therefore, appropriately reducing the acetylation modification level of hemicellulose is an effective method to improve the degradation efficiency of wood biomass and ethanol production.

[0004] Therefore, moderately reducing cell wall acetylation through genetic manipulation, thereby partially weakening the cross-linking between cell wall components and facilitating the removal of lignin and hemicellulose, may be an effective way to improve the degradation and conversion of cell walls into bioethanol. Whether editing the mutant TBL gene using CRISPR / Cas9 technology can genetically improve the cell wall and enhance its enzymatic conversion efficiency and ethanol yield has not been reported. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide an application of editing the ptoESK1 gene (a member of the TBL family) in improving the sugar production efficiency and / or ethanol yield of poplar trees; another objective of the present invention is to provide a method for improving the sugar production efficiency and / or ethanol yield of poplar trees.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. Application of editing the ptoESK1 gene in improving the sugar production efficiency and / or ethanol yield of poplar trees, wherein the nucleic acid sequence of the ptoESK1 gene is shown in SEQ ID No. 5.

[0008] Preferably, the method for editing the ptoESK1 gene in this invention uses CRISPR / Cas9 technology.

[0009] In a preferred embodiment of the present invention, the method for editing the ptoESK1 gene involves transforming a PtoESK1 CRISPR / Cas9 gene editing vector into Populus tomentosa to obtain transgenic plants with PtoESK1 gene editing mutations, resulting in significantly improved sugar production efficiency in the transgenic plants.

[0010] Preferably, in this invention, the CRISPR / Cas9 gene editing vector is formed by double strands of SEQ ID No. 1 and SEQ ID No. 2, and SEQ ID No. 3 and SEQ ID No. 4, which are linked to the pYLCRISPR / Cas9-DH / B vector backbone.

[0011] 2. A method for improving the sugar production efficiency and / or ethanol yield of poplar varieties, comprising transforming a PtoESK1 CRISPR / Cas9 gene editing vector into Populus tomentosa to obtain transgenic plants with PtoESK1 gene editing mutations.

[0012] Preferably, the method for transforming Populus tomentosa is mediated by Agrobacterium.

[0013] Preferably, the Agrobacterium is Agrobacterium GV3101.

[0014] The beneficial effects of this invention are as follows: This invention utilizes CRISPR / Cas9 technology to edit PtoESK1 in Populus tomentosa, resulting in transgenic plants with significantly improved cell wall enzymatic conversion efficiency for sugar production and ethanol yield. This invention lays the foundation for genetic improvement of plant cell walls and targeted molecular breeding, provides new gene resources for plant genetic engineering, and has broad application prospects. Attached Figure Description

[0015] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0016] Figure 1 A schematic diagram illustrating the construction of CRISPR / Cas9 editing vectors;

[0017] Figure 2 Image showing the results of editing CRISPR / Cas9 transgenic plants;

[0018] Figure 3 The graph shows the cell wall enzymatic hydrolysis conversion efficiency and ethanol yield determination of the stems of transgenic plants. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0020] Example 1: Construction of CRISPR / Cas9 editing vector

[0021] Based on the 1473bp CDS sequence length of PtoESK1 (SEQ ID No. 5), two target sites were designed at exon 1. Figure 1 The primers are shown below. Two rounds of nested PCR were used to amplify and construct sgRNA expression cassettes containing the target sequences. After digestion with BsaI, the two target sites were ligated into the pUC18 vector containing the AtU3b and AtU3d promoters, respectively. Using the GoldenGate cloning strategy (Engler et al., 2008; 2009), the pUC18 and pYLCRISPR / Cas9P35S vectors were digested with BsaI, ligating simultaneously to assemble the two sgRNA expression cassettes onto the pYLCRISPR / Cas9P35S-H vector backbone. A schematic diagram of the target sites is attached. Figure 1 As shown.

[0022] Target 1, forward primer: 5'-gtcaTTCTTGAACCAACCGAAGCT-3' (SEQ ID No. 1), reverse primer: 5'-aaacAGCTTCGGTTGGTTCAAGAA-3' (SEQ ID No. 2);

[0023] Target 2, forward primer: 5'-gtcaTCAAACTCCTGATCAACTTG-3' (SEQ ID No. 3), reverse primer: 5'-aaacCAAGTTGATCAGGAGTTTGA-3' (SEQ ID No. 4).

[0024] Example 2: Genetic transformation of Populus tomentosa

[0025] In this study, wild-type Populus tomentosa was used for genetic transformation, and Agrobacterium-mediated leaf disc infection was employed for the transformation.

[0026] 1) Culture of Agrobacterium

[0027] Agrobacterium GV3101 containing the recombinant plasmid was inoculated onto YEP solid medium containing the corresponding antibiotic and cultured at 28°C; single clones were picked and inoculated onto YEP liquid medium (containing the corresponding antibiotic) and cultured at 28°C until OD500. 600 The concentration should be 0.6–0.8; transfer a live bacterial culture to fresh YEP medium at a ratio of 1:100 and incubate at 28°C until the OD value reaches 0.6–0.8. 600Centrifuge at 0.6–0.8 °C at 4 °C to collect the bacterial cells, resuspend them in 30 mL of WPM liquid medium supplemented with acetylsyl syringone, and incubate in a shaker at 28 °C for 1 h.

[0028] 2) Agrobacterium-mediated leaf disc infection

[0029] Take sterile leaves from tissue culture seedlings and cut them into 0.5 × 0.5 cm pieces on a clean bench. 2 Place the leaf discs of the desired size into the pre-suspended Agrobacterium bacterial solution and immerse them for 10 minutes. Gently shake the bacterial solution every 2-3 minutes to ensure that the leaf discs are fully immersed.

[0030] 3) Co-cultivation of Populus tomentosa

[0031] The infected leaf discs were removed with sterilized tweezers, placed on sterilized filter paper, and the bacterial solution was blotted dry. The leaf discs were then laid flat on WPM co-culture medium and incubated in the dark at 25°C for 2 days.

[0032] 4) Selection and cultivation of Populus tomentosa

[0033] After co-culturing for 2 days, the transformed explants were transferred to a selective medium that can induce callus and cultured at 25°C in the dark for 3–5 weeks, with the selective medium being replaced approximately every 5 days.

[0034] 5) Populus tomentosa bud induction culture

[0035] When white, loose callus tissue appears around the leaf margin, transfer the callus tissue to WPM budding medium on a clean bench and culture it at 25°C under light for about 4 to 5 weeks, replacing the WPM budding medium every 10 days.

[0036] 6) Rooting culture of Populus tomentosa

[0037] When the adventitious buds grow to about 5 cm, they are transferred to WPM rooting medium containing the corresponding antibiotics to induce rooting.

[0038] 7) Transplanting of Populus tomentosa

[0039] When the seedlings have a well-developed root system, remove them, rinse off the agar from the roots, transplant them into a greenhouse for cultivation, and cover them with plastic wrap to keep them warm and moist.

[0040] WPM resuspension: WPM powder + 2.14 g + 30 g sucrose + 100 μmol AS;

[0041] WPM co-culture medium: WPM powder + 2.14 g + 30 g sucrose + 100 μmol AS + 1.0 mg NAA + 2.0 mg ZT;

[0042] WPM selective medium: WPM powder + 2.14 g + 30 g sucrose + 9 mg Hyg + 1.0 mg NAA + 2.0 mg ZT + 400 mg Cef;

[0043] WPM germination medium: WPM powder + 2.14 g + 30 g sucrose + 9 mg Hyg + 0.1 mg NAA + 2.0 mg ZT + 400 mg Cef;

[0044] WPM rooting medium: WPM + 30 g sucrose + 9 mg Hyg + 0.1 mg NAA + 400 mg Cef;

[0045] YEP medium: 10 g yeast extract, 10 g peptone, 5 g NaCl, pH 7.0, autoclaved at 121 ℃ for 20 minutes. For solid medium, add 10-12 g agar powder before sterilization.

[0046] LB medium: 5 g yeast extract, 10 g peptone, 10 g NaCl, pH = 7.0, autoclaved at 121 ℃ for 20 min. For solid medium, add 10-12 g agar powder before sterilization.

[0047] Example 3: Identification of transgenic positive seedlings

[0048] To screen for successfully knocked-out transgenic lines, specific primers were designed to amplify the genomic DNA in the transgenic material, and the results were then sequenced. The results are attached. Figure 2 As shown, mutations occurred in both strains 19 and 25. In strain 19, one base was inserted into the first target site, and in strain 25, one base was inserted into the second target site. These base insertions led to frameshift mutations in the gene.

[0049] Example 4: Determination of stem degradation and transformation efficiency of transgenic plants

[0050] NaOH pretreatment and enzymatic hydrolysis:

[0051] 1) Weigh 0.3000 g of straw powder into a 15 mL centrifuge tube, add 10 mL of distilled water, treat at 50℃ and 150 r / min for 2 h, centrifuge at 3000 g for 5 min, and wash the residue three times with distilled water to remove soluble sugars.

[0052] 2) Add 6 mL of 4% (w / v) NaOH solution, incubate at 50℃ and 150 r / min for 2 h. Perform three replicates for each sample.

[0053] 3) After the treatment is completed, centrifuge the sample at 3000 ×g for 5 min, take 1 mL of the pretreatment solution, dilute it by a certain factor, and determine the content of C6 and C5 sugars;

[0054] 4) Remove the remaining supernatant, add 10 mL of distilled water to wash, centrifuge at 3000 ×g for 5 min, and discard the supernatant; repeat the washing of residue 6 times, and finally check the pH to ensure that it is washed to neutral.

[0055] 5) The residue was washed once with 10 mL of 0.2 mol / L pH 4.8 acetate buffer, then 3 mL of 3.2 g / L cellulose complex enzyme solution was added, and the volume was adjusted to 6 mL with pH 4.8 acetate buffer, resulting in a final enzyme concentration of 1.6 g / L. The mixture was then placed in a shaker and incubated at 150 r / min and 50℃ for 48 h. One mL of the enzymatic hydrolysate was taken, diluted a certain factor, and the C6 and C5 sugar contents were determined. The results are as follows: Figure 3 As shown in Figure A, the results indicate that NaOH pretreatment increased the efficiency of hexose (C6) production by the ESK1-edited transgenic lines by 12%-26% compared to the wild-type control.

[0056] H2SO4 pretreatment and enzymatic hydrolysis:

[0057] 1) Weigh 0.3000 g of straw powder into a 15 mL centrifuge tube, add 10 mL of distilled water, treat at 50℃ and 150 r / min for 2 h, centrifuge at 3000 ×g for 5 min, and wash the residue with distilled water 3 times to remove soluble sugars.

[0058] 2) Add 6 mL of 4% (v / v) H2SO4 solution, treat at 120℃ for 20 min; cool the sample to room temperature and then treat at 50℃, 150 r / min for 2 h; each sample has 3 replicates;

[0059] 3) After the treatment is completed, centrifuge the sample at 3000 ×g for 5 min, take 1 mL of the pretreatment solution, dilute it by a certain factor, and determine the content of C6 and C5 sugars;

[0060] Remove the remaining supernatant, add 10 mL of distilled water to wash, centrifuge at 3000 ×g for 5 min, and discard the supernatant; repeat the washing of residue 6 times, and finally check the pH to ensure that it is washed to neutral.

[0061] 5) The residue was washed once with 10 mL of 0.2 mol / L pH 4.8 acetate buffer, then 3 mL of 3.2 g / L cellulose complex enzyme solution was added, and the volume was adjusted to 6 mL with pH 4.8 acetate buffer, resulting in a final enzyme concentration of 1.6 g / L. The solution was then placed in a shaker and incubated at 150 r / min and 50℃ for 48 h. One mL of the enzymatic hydrolysate was taken, diluted a certain factor, and the C6 sugar content was determined. The results are as follows: Figure 3 As shown in Figure A, the results indicate that after H2SO4 pretreatment, the efficiency of hexose (C6) production by the ESK1-edited transgenic lines increased by more than 18% compared with the wild-type control.

[0062] Colorimetric determination of C6 and C5 sugars

[0063] C6 determination

[0064] 1) Preparation of glucose standard solution

[0065] Weigh 100.00 mg of glucose dried to constant weight, dissolve it in water and bring the volume to 100 mL, mix well, and prepare a standard solution of 1.00 mg / mL.

[0066] 2) Creating a standard curve

[0067] Take 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL of 1.00 mg / mL glucose standard solution into 100 mL volumetric flasks, dilute to volume with water, and then take 1.0 mL of each of the above solutions into 10 mL stoppered glass test tubes. Add 2.0 mL of anthrone reagent (0.2 g anthrone dissolved in 100.0 mL concentrated sulfuric acid), shake quickly, heat in boiling water for 5 min, cool with tap water, and measure color at 620 nm. Use 1.0 mL of distilled water as a blank sample.

[0068] C5 determination

[0069] 1) Preparation of xylose standard solution

[0070] Weigh 100.00 mg of xylose dried to constant weight, dissolve it in water and bring the volume to 100 mL, then mix well to form a standard solution of 1.00 mg / mL.

[0071] 2) Creating a standard curve

[0072] Take 0.5 mL, 1.0 mL, 2.0 mL, 3.0 mL, and 4.0 mL of 1.00 mg / mL xylose standard solution into 100 mL volumetric flasks, and dilute to volume with water. Then, take 1.0 mL of each of the above solutions into 10 mL stoppered glass test tubes. First, add 134 µL of reagent A (prepared by dissolving 6 g of phenol in 100.0 mL of ethanol), then add 2.0 mL of reagent B (prepared by dissolving 0.1 g of FeCl3 in 100 mL of 37% concentrated hydrochloric acid). Mix well, heat in boiling water for 20 min, cool to room temperature with tap water, and measure the color at a wavelength of 660 nm using a spectrophotometer. Use 1.0 mL of distilled water as a blank sample.

[0073] 3) Dilute the supernatant obtained above with distilled water 10 times, take an appropriate volume (less than 1 mL) of the diluted sample, add water to 1.0 mL, and perform colorimetric analysis according to the method for preparing the standard curve (so that the reading is between 0.2 and 0.8).

[0074] Example 5: Determination of bioethanol yield from stems of transgenic plants

[0075] 1. Biomass fermentation

[0076] After pretreatment, the supernatant was adjusted to pH 4.8, and cellulose complex enzyme (final concentration 1.6 g / L) was added for 48 h. Under aseptic conditions (clean bench), Angel thermoresistant yeast (final concentration 0.5 g / L) was added to the mixture of enzymatic hydrolysis supernatant and residue, and the mixture was incubated at 37˚C for 48 h. After fermentation, the entire fermentation system (fermentation broth and residue) was transferred to a 250 mL round-bottom flask, and distilled water was added to bring the fermentation broth to 50.0 mL. The heating mantle temperature was set to 105˚C, and the first 20.0 mL of distillate was collected. The ethanol content was measured, and the ethanol yield was calculated.

[0077] 2. Ethanol content determination

[0078] Ethanol content determination: The ethanol content was determined according to the potassium dichromate method (Li et al 2014).

[0079] 1) Preparation of standard curve

[0080] Take 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, and 3.0 mL of anhydrous ethanol from the standard solution into 100 mL volumetric flasks, respectively, and dilute to volume with water. Then, take 1.0 mL of each of the above solutions into 10 mL stoppered glass test tubes, add 2.0 mL of 5% K₂Cr₂O₇, mix well, and boil in a water bath for 10 min, then cool rapidly. Add 7.0 mL of distilled water, mix well, and measure the color at a visible wavelength of 600 nm, using 1.0 mL of distilled water as a control.

[0081] 2) Determination of ethanol content in fermentation broth

[0082] After diluting the above sample according to a certain ratio, take 1.0 mL into a 10.0 mL colorimetric tube, add 2.0 mL of 5% K₂Cr₂O₇, mix well, and boil in a water bath for 10 min, then cool rapidly. Add 7.0 mL of distilled water, mix well, and measure the color at a visible wavelength of 600 nm. Use 1.0 mL of distilled water as a control. The results are as follows. Figure 3 As shown in B.

[0083] The results showed that the saccharification efficiency of the stem cell walls of the PtoESK1-KO transgenic material was significantly higher than that of the wild type under both pretreatments. Pretreatment with NaOH and H2SO4 increased the enzymatic saccharification efficiency by 12%-26% and 18%-19%, respectively. Figure 3 (A). As the final product of biomass utilization, ethanol yield is an important indicator for measuring biomass conversion and utilization, and also a crucial factor in determining whether biomass ethanol can be commercially produced. Therefore, the yield of cellulose ethanol produced by transgenic materials was further compared. The ethanol yield of PtoESK1-KO transgenic material was significantly higher than that of the wild-type control material under both pretreatments, with an increase of 10%–32% under NaOH treatment and 29%–31% under H2SO4 treatment. Figure 3 (B). These results indicate that the PtoESK1-KO transgenic material can produce more cellulose ethanol, increasing wood utilization efficiency.

[0084] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of ptoESK1 gene knockout in improving hexose production efficiency and / or ethanol yield in poplar trees, characterized by: The nucleic acid sequence of the ptoESK1 gene is shown in SEQ ID No.

5.

2. The application according to claim 1, characterized in that: The method for knocking out the ptoESK1 gene uses CRISPR / Cas9 technology.

3. The application according to claim 1, characterized in that: The method for knocking out the ptoESK1 gene involves transforming a PtoESK1 CRISPR / Cas9 gene editing vector into Populus tomentosa to obtain transgenic plants with a PtoESK1 gene mutation. The transgenic plants obtained have a significantly improved hexose production efficiency.

4. The application according to claim 3, characterized in that: The CRISPR / Cas9 gene editing vector consists of a double strand formed by SEQ ID No. 1 and SEQ ID No. 2, and SEQ ID No. 3 and SEQ ID No. 4, which is linked to the pYLCRISPR / Cas9-DH / B vector backbone.

5. A method for improving the efficiency of poplar in producing hexose and / or ethanol, characterized in that: The CRISPR / Cas9 gene editing vector of PtoESK1 was transformed into Populus tomentosa to obtain transgenic plants with the ptoESK1 gene knockout mutation; the nucleic acid sequence of the ptoESK1 gene is shown in SEQ ID No.

5.

6. The method according to claim 5, characterized in that: The method for transforming Populus tomentosa is mediated by Agrobacterium.

7. The method according to claim 6, characterized in that: The Agrobacterium is Agrobacterium GV3101.

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