Genetically engineered bacterium capable of efficiently expressing xylanase and application of genetically engineered bacterium in degradation of straw agricultural wastes

Abstract

The invention discloses a genetically engineered bacterium capable of efficiently expressing xylanase and application of the genetically engineered bacterium in degradation of straw agricultural wastes, and belongs to the technical field of bioengineering. By optimizing the expression cassette of the xylanase gene and increasing the expression quantity of the Hac1 gene, the expression quantity of the xylanase is remarkably improved, and the cost of industrial enzyme production is reduced. The successful construction of the engineering bacteria further reduces the cost of degrading straw agricultural wastes to produce xylooligosaccharide, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a genetically engineered bacterium for highly expressing xylanase gene and its application in degrading straw-like agricultural waste. Background Art

[0002] Agricultural wastes, such as corncobs, sugarcane bagasse, cottonseed hulls and rape straws, have long been discarded or burned, causing serious environmental pollution. However, these agricultural wastes are rich in hemicellulose and have great application potential as biomass resources, and can be used to produce high-value biochemical products. In particular, xylan, the main component of hemicellulose, is considered an ideal raw material for preparing xylooligosaccharide (XOS). Xylooligosaccharide has broad application prospects as a prebiotic in the fields of food, medicine, agriculture, etc.

[0003] Xylanase (E.C.3.2.1.8) is a key enzyme for hydrolyzing xylan to produce xylooligosaccharide and plays an important role in the high-value utilization of lignocellulose. Trichoderma reesei is an important strain in the field of biotechnology and has a complete lignocellulose-degrading enzyme system. The xylanase Xyn2 (GenBank accession number: ETR98242.1) secreted by this strain has high hydrolytic activity towards xylan and has been widely used in pulp biobleaching and feed additives. The mutant Xyn2A obtained by introducing a pair of disulfide bonds (T2C / T28C) into Xyn2 significantly improves the enzyme activity and thermal stability of the enzyme at high temperature (65°C). Therefore, the large-scale production of Xyn2A and its degradation and saccharification processes of hemicellulose in agricultural straws have important research significance and application value.

[0004] The lignocellulose in crop straw has a complex structure, and the crosslinking of lignin and the side-chain modification of xylan limit the degradation efficiency of xylanase. Therefore, before hydrolyzing lignocellulose with xylanase, it is often necessary to pretreat lignocellulose. The pure physical method mainly uses steam explosion method. This method makes most of the xylan dissolve in the liquid, but there are also a large number of lignin hydrolysis products and furfural in the solution, etc. The subsequent separation and purification are difficult and large pressure vessels are required. The current pretreatment methods mainly combine physical methods with chemical methods. The materials are often first crushed and sieved by a crusher to improve the efficiency of subsequent chemical treatment. The chemical methods mainly include acid hydrolysis, alkali hydrolysis and hydrogen peroxide oxidation method. Acid hydrolysis makes the hemicellulose dissolve in the acid solution, and it is difficult to separate from the lignin hydrolysis products subsequently, while the hydrogen peroxide method has a high cost in industrial production. In contrast, alkali hydrolysis is highly efficient in lignin removal and can also deacetylate xylan. It is a milder and more economical method, providing an economically feasible way for the high-value utilization of straw-like agricultural waste. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a genetically engineered bacterium that highly expresses xylanase and its application in degrading straw-like agricultural waste.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] The first object of the present invention is to provide a recombinant plasmid pSc048, and the recombinant plasmid Sc048 is constructed by the following method:

[0008] 1) Construct the Xyn2A expression cassette by Golden Gate Cloning, including the FMD promoter, αMF* signal peptide, Xyn2A gene and AOX1 terminator, and load the expression cassette into the backbone plasmid pBK to construct plasmid pSc026;

[0009] 2) Construct the Hac1 expression cassette by Golden Gate Cloning, including the FBA2 promoter, Hac1 gene and DAS1 terminator, and load the expression cassette into the backbone plasmid pBK to construct plasmid pSc035;

[0010] 3) Enzymatically digest and ligate the pSc026 plasmid by the Biobrick method to construct a recombinant plasmid pSc038 containing 2 copies of the Xyn2A expression cassette;

[0011] 4) Enzymatically digest and ligate pSc038 and pSc035 by the Biobrick method to construct a triple-expression-cassette recombinant plasmid pSc048 containing 2 copies of the Xyn2A expression cassette and 1 copy of the Hac1 expression cassette.

[0012] In a specific embodiment, the nucleotide sequence of the FMD promoter is as shown in SEQ ID NO:3.

[0013] In a specific embodiment, the nucleotide sequence of the αMF* signal peptide is as shown in SEQ ID NO:9.

[0014] In a specific embodiment, the nucleotide sequence of the Xyn2A gene is as shown in SEQ ID NO:11.

[0015] In a specific embodiment, the nucleotide sequence of the AOX1 terminator is as shown in SEQ ID NO:17.

[0016] In a specific embodiment, the nucleotide sequence of the FBA2 promoter is as shown in SEQ ID NO:6.

[0017] In a specific embodiment, the nucleotide sequence of the Hac1 gene is as shown in SEQ ID NO:14.

[0018] In a specific embodiment, the nucleotide sequence of the DAS1 terminator is as shown in SEQ ID NO:19.

[0019] The second object of the present invention is to provide a genetically engineered bacterium for highly expressing xylanase, which contains the recombinant plasmid pSc048 described above.

[0020] By introducing the xylanase gene Xyn2A into Pichia pastoris, and by optimizing the promoter, signal peptide of the Xyn2A expression cassette and the copy number of the expression cassette, and at the same time increasing the expression level of the Hac1 gene, a genetically engineered bacterium is obtained.

[0021] The third object of the present invention is to provide a method for constructing the above-mentioned genetically engineered bacterium, and the steps are as follows:

[0022] 1) Construct an Xyn2A expression cassette by Golden Gate cloning, which contains an FMD promoter, an αMF* signal peptide, an Xyn2A gene and an AOX1 terminator, and load the expression cassette into the backbone plasmid pBK to construct plasmid pSc026;

[0023] 2) Construct an Hac1 expression cassette by Golden Gate cloning, which contains an FBA2 promoter, an Hac1 gene and a DAS1 terminator, and load the expression cassette into the backbone plasmid pBK to construct plasmid pSc035;

[0024] 3) Enzymatically digest and ligate the pSc026 plasmid by the BioBrick method to construct a recombinant plasmid pSc038 containing 2 copies of the Xyn2A expression cassette;

[0025] 4) By using the BioBrick method, digest pSc038 and pSc035 with enzymes and ligate them to construct a triple-expression cassette recombinant plasmid pSc048 containing 2 copies of the Xyn2A expression cassette and 1 copy of the Hac1 expression cassette;

[0026] 5) Transform pSc048 into Pichia pastoris X33, and screen for positive transformants on bleomycin plates.

[0027] The fourth object of the present invention is a method for fermenting and producing xylanase using the above recombinant engineering bacteria, and the method comprises the following steps: using BSM as the fermentation medium, after inoculating at an inoculation amount of 10%, after the glucose in the medium is consumed, add 70% glucose containing 12 mL / L PTM1 by exponential fed-batch, culture the bacteria until the OD 600 reaches 200, and then induce the expression of xylanase by feeding methanol containing 12 mL / L PTM1, and maintain the concentration of methanol at 2.4 g / L for induction expression.

[0028] In a specific embodiment, the composition of the BSM medium is as follows: 85% H3PO4 26.7 mL / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glucose 20 g / L, PTM1 4.4 mL / L.

[0029] In a more specific embodiment, the composition of PTM1 is as follows: CuSO4·5H2O 6 g / L, KI 0.08 g / L, MnSO4·H2O 3 g / L, MoNa2O4 0.2 g / L, H3BO3 0.02 g / L, CoCl2 0.5 g / L, ZnCl2 20 g / L, FeSO4·7H2O 65 g / L, Biotin 0.2 g / L, H2SO4 5 mL / L.

[0030] The fifth object of the present invention is to protect the xylanase prepared by the aforementioned method.

[0031] The sixth object of the present invention is the application of the aforementioned xylanase in the degradation of straw-like agricultural waste to produce xylo-oligosaccharides, especially the degradation of corncobs, bagasse, cottonseed hulls and rape straws.

[0032] The specific steps are as follows: add pretreated straw to the sodium acetate buffer solution at pH 5.0 so that the concentration of straw powder in the reaction system is 100 g / L, and the addition amount of xylanase Xyn2A is 1 mg / g straw powder, and react at 50 °C and a stirring speed of 200 rpm for 12 h.

[0033] The seventh object of the present invention is to use the sugar solution obtained by degrading straw-like agricultural waste to promote plant growth, especially the growth promotion of cucumber seedlings and tomato seedlings.

[0034] Beneficial effects

[0035] The present invention aims to provide a genetically engineered bacterium that highly expresses xylanase and its application in degrading straw-like agricultural waste. Compared with the prior art, it has the following beneficial effects:

[0036] (1) The recombinant genetically engineered bacterium pSc048-X33 of the present invention can highly express xylanase Xyn2A. At the 5L fermenter level, the content of Xyn2A in the supernatant reaches 10.2 g / L, and the enzyme activity is as high as 45100 U / mL;

[0037] (2) The present invention uses the Xyn2A fermentation supernatant to treat agricultural waste. At a relatively low enzyme dosage (1 g of material: 1 mg of Xyn2A), and under the condition that the solid-liquid ratio of the enzymatic hydrolysis reaction is 1:10, the reducing sugar content in the supernatant reaches 9.0 - 31.1 mg / mL.

[0038] (3) The sugar solution obtained by degrading agricultural waste in the present invention can significantly promote the growth of cucumber seedlings and tomato seedlings. Spraying cucumber seedlings and tomato seedlings with sugar concentrations of 2400 ppm and 100 ppm respectively, the fresh weight of the above-ground parts of the plants increases by 34% and 49% respectively. Description of the drawings

[0039] Figure 1 Schematic diagram for the construction of the Pichia pastoris expression toolkit in Example 1;

[0040] Figure 2 For Example 2, the xylanase activity in the supernatant after 72 h of shake flask-induced fermentation of the recombinant engineering bacterium;

[0041] Figure 3 For Example 3, the induction fermentation time curve of the pSc048-X33 recombinant bacterium in a 5L fermenter;

[0042] Figure 4 For Example 3, the SDS-PAGE results of the protein of the recombinant xylanase expressed in a 5L fermenter; Lane 1 is the protein marker, and Lanes 2 - 11 are the extracellular proteins induced and expressed for 0, 12, 24, 36, 48, 60, 72, 84, 96, and 108 h respectively;

[0043] Figure 5 For Example 3, the results of the two obvious bands in SDS-PAGE retrieved by time-of-flight mass spectrometry combined with Mascot software. (A) Results of Band I; (B) Results of Band II;

[0044] Figure 6 For the cucumber seedling phenotype in Example 8;

[0045] Figure 7 For the physiological indexes of cucumber seedlings in Example 8;

[0046] Figure 8 For the tomato seedling phenotype in Example 9;

[0047] Figure 9 For the physiological indexes of tomato seedlings in Example 9; Specific implementation manners

[0048] The present invention will be further described in detail below in conjunction with embodiments. Reagents or instrument and equipment not indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0049] Construction of the Pichia pastoris expression toolkit in Example 1

[0050] Based on the seamless connection principle of Golden Gate Cloning, a toolkit for the Pichia pastoris expression system was constructed using Type IIS enzymes. As Figure 1 shown, this toolkit consists of three levels of plasmids: Level 0, Level 1, and Level 2.

[0051] 1. Construction of the Level 0 element plasmid

[0052] The Level 0 plasmid contains specific functional elements, including a promoter, a signal peptide, a target gene, and a terminator. These elements are loaded into the backbone plasmid pYTK001 (product of Addgene, catalog number #65108) through BsmBI-mediated Golden Gate Cloning. The promoter, signal peptide, target gene, and terminator used were synthesized by Anhui General Company and loaded into the pUC57 plasmid. The reaction system used for the construction of the Level 0 plasmid is as follows: 10 fmol of the pYTK001 plasmid, 10 fmol of the pUC57 plasmid containing the functional elements, 1 μL of T4 DNA Ligase (NEB), 1 μL of BsmBI (NEB), 2 μL of T4 DNA ligase buffer, and ddH2O was added to make up the volume to 20 μL. The above system was mixed well, and an assembly reaction was carried out using a PCR instrument. The program is as follows:

[0053]

[0054] 8 μL of the assembly product was taken and transformed into Escherichia coli, and the sequence was verified by sequencing to obtain the Level 0 element plasmid with the correct sequence.

[0055] 2. Construction of the Level 1 single expression cassette plasmid

[0056] Level 1 is a single expression cassette plasmid. Through BsaI-mediated Golden Gate assembly, specific Level 0 plasmid is loaded into the backbone plasmid pBK. The reaction system used is as follows: 10 fmol of pBK plasmid, 10 fmol of Level 0 element plasmid, 1 μL of T4 DNA Ligase (NEB), 1 μL of BsaI (NEB), 2 μL of T4 DNA ligase buffer, and ddH2O is used to make up the volume to 20 μL. Mix the above system evenly, and use a PCR instrument to carry out the assembly reaction. The procedure is as follows:

[0057]

[0058] Take 8 μL of the assembly product to transform Escherichia coli, and sequence verification is carried out to obtain the Level 1 element plasmid with correct sequence.

[0059] 3. Construction of Level 2 multi-expression cassette plasmid

[0060] Level 2 plasmid conducts tandem of expression cassettes through BioBrick method mediated by EcoRI, XbaI, SpeI and PstI. All the plasmids constructed are as follows:

[0061]

[0062]

[0063] Example 2 Construction and expression of Pichia pastoris recombinant strain of Xyn2A

[0064] 1. Construction of recombinant strain

[0065] The plasmids pSc020, pSc021, pSc022, pSc023, pSc024, pSc025, pSc026, pSc027, pSc038, pSc039, pSc040, pSc041, pSc042, pSc043, pSc044, pSc045, pSc046, pSc047, pSc048, pSc049, pSc050 in Example 1 were linearized by the restriction endonuclease KpnI and then electrotransformed into the expression host P. pastoris X33. The transformed bacterial suspension was spread on a plate containing bleomycin, and positive transformants were screened by resistance and named recombinant bacteria pSc020-X33, pSc021-X33, pSc022-X33, pSc023-X33, pSc024-X33, pSc025-X33, pSc026-X33, pSc027-X33, pSc038-X33, pSc039-X33, pSc040-X33, pSc041-X33, pSc042-X33, pSc043-X33, pSc044-X33, pSc045-X33, pSc046-X33, pSc047-X33, pSc048-X33, pSc049-X33, pSc050-X33 respectively.

[0066] 2. Shake flask fermentation

[0067] The above recombinant strains were subjected to shake flask fermentation. Single colonies were picked and inoculated into 25 mL of BMGY (yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 12 g / L, YNB 1.34 g / L, glucose 10 g / L) and cultured at 30 °C and 200 rpm for 20 h. The cells were collected by centrifugation, and an appropriate amount of cells was transferred to 100 mL of BMMY (yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 12 g / L, YNB 1.34 g / L) medium. The initial OD600 in the shake flask was controlled to be 2.0, and 1 mL of methanol was added for induction. Thereafter, 1 mL of methanol was added every 24 h for 72 h of induced expression. The xylanase activity and protein content in the supernatant were measured. Definition of xylanase activity unit: Under the conditions of pH 5.0 and 60 °C, the amount of enzyme required to release 1 μmol of reducing sugar (xylose equivalent) from xylan per minute is one enzyme activity unit. The protein content in the fermentation supernatant was measured by the Bradford method.

[0068] The results are as Figure 2 shown. After 72 h of induced expression of the pSc048-X33 strain, the xylanase activity in the supernatant was the highest, reaching 1400 U / mL.

[0069] Example 3 Induced Expression of Recombinant Strain pSc048-X33 in a 5L Fermenter

[0070] Pick 4 monoclonal colonies and inoculate them into 4 bottles of 50 mL YPD medium (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) and culture at 30 °C and 200 rpm for 18 h. Transfer them to a 5L fermenter containing 1800 mL BSM medium (26.7 mL / L of 85% H3PO4, 0.93 g / L CaSO4, 18.2 g / L K2SO4, 14.9 g / L MgSO4·7H2O, 4.13 g / L KOH, 20 g / L glucose, 4.4 mL / L PTM1) at an inoculation amount of 10%. The fermentation parameters are: temperature 30 °C, stirring speed 300 rpm, aeration rate 1 vvm, pH 5.0. Culture until the glucose in the fermenter is completely consumed, and then feed 70% glucose containing 12 mL / L PTM1 (6 g / L CuSO4·5H2O, 0.08 g / L KI, 3 g / L MnSO4·H2O, 0.2 g / L MoNa2O4, 0.02 g / L H3BO3, 0.5 g / L CoCl2, 20 g / L ZnCl2, 65 g / L FeSO4·7H2O, 0.2 g / L Biotin, 5 mL / L H2SO4) into the fermenter in an exponential feeding manner. The feeding volume at different times is as follows in the table:

[0071]

[0072] After the feeding is completed and the cell OD600 reaches 200, start the induced expression. Feed methanol containing 12 mL / L PTM1 to maintain the methanol concentration in the fermenter at 2.4 g / L. The methanol concentration is fed through an on-line methanol detection and feeding controller, and the induced expression lasts for 108 h. Regularly take samples during this period to measure OD 600 , protein concentration and xylanase activity. The results are as Figure 3 shown. After 108 h of induction, OD 600 reaches 550, the protein concentration in the supernatant reaches 10.2 g / L, and the enzyme activity in the supernatant reaches 45100 U / mL. The fermentation broth is detected by SDS-PAGE protein electrophoresis, and the results are as Figure 4 shown. Due to glycosylation differences, Xyn2A shows two distinct bands on the SDS-PAGE diagram, which are 22 kDa and 18 kDa respectively. Perform time-of-flight mass spectrometry on the two bands and compare and retrieve them through Mascot. The results are as Figure 5 shown. Both bands are Xyn2A, and the molecular weight difference is due to partial glycosylation of Xyn2A.

[0073] Example 4 Direct Hydrolysis of Corncobs Using Fermentation Supernatant

[0074] Cut corncobs (cellulose 39.5%, hemicellulose 33.9%, lignin 10.7%) into 10 mm lengths, place them in a high-speed grinder for pulverization, and pass the powder through a 20-mesh sieve. Weigh 200 g of the above-mentioned 20-mesh corncobs and place them in a 5 L beaker. Add 2% KOH solution according to a solid-liquid ratio of 1:10 and soak at 25 °C for 24 h. Add an appropriate amount of 5% dilute sulfuric acid for neutralization, filter out the liquid through 8 layers of gauze, rinse and collect the pretreated corncobs, dry them at 70 °C and weigh them to be 139.2 g. Measure the contents of cellulose, hemicellulose, and lignin in the pretreated corncobs, which are 57.9%, 33.4%, and 3.6% respectively. Take 100 g of the above-mentioned pretreated corncobs and place them in an enzymatic hydrolysis tank. Add pH 5.0, 50 mM sodium acetate buffer according to a solid-liquid ratio of 1:10 and simultaneously add 10 mL of fermentation supernatant containing xylanase. Set the stirring speed at 200 rpm and the temperature at 50 °C, and react for 12 h. After the reaction, the reducing sugar content in the enzymatic hydrolysate is 20.2 mg / mL. Filter the enzymatic hydrolysate, and the mass of the dried residue is 73.4 g. The contents of cellulose, hemicellulose, and lignin are 77.2%, 8.1%, and 4.3% respectively. The degradation rate of hemicellulose in the pretreated corncobs after enzymatic hydrolysis is 82.2%.

[0075] Example 5 Direct Hydrolysis of Rapeseed Straw Using Fermentation Supernatant

[0076] Cut rapeseed straw (cellulose 40.2%, hemicellulose 22.3%, lignin 16.2%) into 10 mm lengths, place them in a high-speed grinder for pulverization, and pass the powder through a 60-mesh sieve. Weigh 200 g of the above-mentioned 60-mesh rapeseed straw and place them in a 5 L beaker. Add 1% KOH solution according to a solid-liquid ratio of 1:10 and soak at 25 °C for 36 h. Add an appropriate amount of 5% dilute sulfuric acid for neutralization, filter out the liquid through 8 layers of gauze, rinse and collect the pretreated rapeseed straw, dry them at 70 °C and weigh them to be 148.3 g. Measure the contents of cellulose, hemicellulose, and lignin in the pretreated rapeseed straw, which are 46.5%, 28.5%, and 17.1% respectively. Take 100 g of the above-mentioned pretreated rapeseed straw and place them in an enzymatic hydrolysis tank. Add pH 5.0, 50 mM sodium acetate buffer according to a solid-liquid ratio of 1:10 and simultaneously add 10 mL of fermentation supernatant containing xylanase. Set the stirring speed at 200 rpm and the temperature at 50 °C, and react for 12 h. After the reaction, the reducing sugar content in the enzymatic hydrolysate is 16.2 mg / mL. Filter the enzymatic hydrolysate, and the mass of the dried residue is 78.7 g. The contents of cellulose, hemicellulose, and lignin are 55.5%, 15.5%, and 20.2% respectively. The degradation rate of hemicellulose in the pretreated rapeseed straw after enzymatic hydrolysis is 57.2%.

[0077] Example 6 Direct hydrolysis of sugarcane bagasse using fermentation supernatant

[0078] Put sugarcane bagasse (cellulose 53.5%, hemicellulose 19.5%, lignin 16.2%) into a high-speed pulverizer for pulverization, and the powder passes through a 20-mesh sieve. Weigh 200 g of the above-mentioned 20-mesh sugarcane bagasse and place it in a 5 L beaker. Add 2% KOH solution at a solid-liquid ratio of 1:10 and soak it at 25 °C for 24 h. Add an appropriate amount of 5% dilute sulfuric acid for neutralization, filter out the liquid through 8 layers of gauze, rinse and collect the pretreated sugarcane bagasse, and dry it at 70 °C. The weight after drying is 153.6 g. Measure the contents of cellulose, hemicellulose and lignin in the pretreated sugarcane bagasse, which are 54.5%, 15.1% and 15.8% respectively. Take 100 g of the above-mentioned pretreated sugarcane bagasse and place it in an enzymatic hydrolysis tank. Add pH 5.0, 50 mM sodium acetate buffer at a solid-liquid ratio of 1:15 and simultaneously add 10 mL of fermentation supernatant containing xylanase. Set the stirring speed at 200 rpm and the temperature at 50 °C, and react for 12 h. After the reaction, the reducing sugar content in the enzymatic hydrolysate is 9.0 mg / mL. The mass of the filter residue after filtering the enzymatic hydrolysate after drying is 76.6 g, and the contents of cellulose, hemicellulose and lignin are 50.1%, 7.2% and 10.1% respectively. The degradation rate of hemicellulose in the pretreated sugarcane bagasse after enzymatic hydrolysis is 63.2%.

[0079] Example 7 Direct hydrolysis of cottonseed hulls using fermentation supernatant

[0080] Put cottonseed hulls (cellulose 36.6%, hemicellulose 27.2%, lignin 19.5%) into a high-speed pulverizer for pulverization, and the powder passes through a 20-mesh sieve. Weigh 200 g of the above-mentioned 20-mesh cottonseed hulls and place it in a 5 L beaker. Add 1% KOH solution at a solid-liquid ratio of 1:10 and soak it at 25 °C for 24 h. Add an appropriate amount of 5% dilute sulfuric acid for neutralization, filter out the liquid through 8 layers of gauze, rinse and collect the pretreated cottonseed hulls, and dry it at 70 °C. The weight after drying is 163.2 g. Measure the contents of cellulose, hemicellulose and lignin in the pretreated cottonseed hulls, which are 43.5%, 29.8% and 17.0% respectively. Take 100 g of the above-mentioned pretreated cottonseed hulls and place it in an enzymatic hydrolysis tank. Add pH 5.0, 50 mM sodium acetate buffer at a solid-liquid ratio of 1:10 and simultaneously add 10 mL of fermentation supernatant containing xylanase. Set the stirring speed at 200 rpm and the temperature at 50 °C, and react for 12 h. After the reaction, the reducing sugar content in the enzymatic hydrolysate is 31.1 mg / mL. The mass of the filter residue after filtering the enzymatic hydrolysate after drying is 64.5 g, and the contents of cellulose, hemicellulose and lignin are 56.3%, 7.5% and 26.2% respectively. The degradation rate of hemicellulose in the pretreated cottonseed hulls after enzymatic hydrolysis is 83.8%.

[0081] Example 8 Foliar spraying of cucumber seedlings with hydrolyzed sugar solution

[0082] The hydrolyzed sugar solution was appropriately diluted to make the reducing sugar concentration reach 2.4 mg / mL as the sugar solution for spraying. Cucumber seedlings at the three-leaf stage (variety: Lufeng) were transplanted into 300 g of substrate soil, and the sugar solution was sprayed on the leaves of the cucumber seedlings. 5 mL was sprayed on each seedling, and the spraying was carried out once every 4 days for a total of three times, with deionized water as the control. After three sprays, the phenotypes were as Figure 1 shown. Compared with the control group, the plant height, root length, above-ground fresh weight, and root fresh weight of the treatment group increased by 32%, 28%, 34%, and 60% respectively. The physiological indexes were as Figure 2 shown. Compared with the control group, the content of the root growth hormone IAA in the treatment group increased by 45%, the leaf photosynthesis rate increased by 51%, and the intercellular carbon dioxide concentration in the leaves decreased by 7%.

[0083] Example 9 Spraying the leaves of tomato seedlings with the hydrolyzed sugar solution

[0084] The hydrolyzed sugar solution was appropriately diluted to make the reducing sugar concentration reach 0.1 mg / mL as the sugar solution for spraying. Tomato seedlings at the three-leaf stage (variety: Micro Tom) were transplanted into 300 g of substrate soil, and the sugar solution was sprayed on the leaves of the cucumber seedlings. 5 mL was sprayed on each seedling, and the spraying was carried out once every 4 days for a total of three times, with deionized water as the control. After three sprays, the phenotypes were as Figure 3 shown. Compared with the control group, the plant height, root length, above-ground fresh weight, and root fresh weight of the treatment group increased by 34%, 52%, 49%, and 120% respectively. The physiological indexes were as Figure 4 shown. Compared with the control group, the content of the root growth hormone IAA in the treatment group increased by 40%, the leaf photosynthesis rate increased by 80%, and the intercellular carbon dioxide concentration in the leaves decreased by 11%. The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are used as the protection scope.

Claims

1. A recombinant plasmid pSc048, which is constructed by the following method: 1) Construct an Xyn2A expression cassette by Golden Gate cloning, which contains an FMD promoter, an αMF* signal peptide, the Xyn2A gene, and an AOX1 terminator, and load the expression cassette into the backbone plasmid pBK to construct plasmid pSc026; 2) Construct an Hac1 expression cassette by Golden Gate cloning, which contains an FBA2 promoter, the Hac1 gene, and a DAS1 terminator, and load the expression cassette into the backbone plasmid pBK to construct plasmid pSc035; 3) Use the BioBrick method to digest and ligate the pSc026 plasmid to construct a recombinant plasmid pSc038 containing 2 copies of the Xyn2A expression cassette; 4) Use the BioBrick method to digest and ligate pSc038 and pSc035 to construct a triple-expression cassette recombinant plasmid pSc048 containing 2 copies of the Xyn2A expression cassette and 1 copy of the Hac1 expression cassette.

2. The recombinant plasmid pSc048 according to claim 1, wherein the nucleotide sequence of the Xyn2A gene is as shown in SEQ ID NO:11; preferably, the nucleotide sequence of the FMD promoter is as shown in SEQ ID NO:3; preferably, the nucleotide sequence of the αMF* signal peptide is as shown in SEQ ID NO:9; preferably, the nucleotide sequence of the AOX1 terminator is as shown in SEQ ID NO:

17.

3. The recombinant plasmid pSc048 according to claim 1, wherein the nucleotide sequence of the Hac1 gene is as shown in SEQ ID NO:14; preferably, the nucleotide sequence of the FBA2 promoter is as shown in SEQ ID NO:6; preferably, the nucleotide sequence of the DAS1 terminator is as shown in SEQ ID NO:

19.

4. A genetically engineered bacterium for highly expressing xylanase, which contains the recombinant plasmid pSc048 according to any one of claims 1-3.

5. The method for constructing the genetically engineered bacterium according to claim 4, characterized in that, The construction method comprises the following steps: Step 1, construct the recombinant plasmid pSc048 according to any one of claims 1-3; Step 2, transform the pSc048 constructed in Step 1 into Pichia pastoris X33, and screen for positive transformants by a bleomycin plate.

6. A method for fermenting and producing xylanase using the recombinant engineering bacterium described in claim 4, the method comprising the following steps: using BSM as a fermentation medium, inoculating the recombinant engineering bacterium described in claim 4 at an inoculation amount of 10%, and after the glucose in the medium is consumed, adding 70% glucose containing 12 mL / L PTM1 by exponential feeding, culturing the bacterial cells until OD 600 reaches 200, and then inducing the expression of xylanase by feeding methanol containing 12 mL / L PTM1, maintaining the concentration of methanol at 2.4 g / L, and inducing expression; Preferably, the composition of the BSM medium is as follows: 85% H3PO4 26.7 mL / L, CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glucose 20 g / L, PTM1 4.4 mL / L; Preferably, the PTM1 is composed of: 6 g / L of CuSO4·5H2O, 0.08 g / L of KI, 3 g / L of MnSO4·H2O, 0.2 g / L of MoNa2O4, 0.02 g / L of H3BO3, 0.5 g / L of CoCl2, 20 g / L of ZnCl2, 65 g / L of FeSO4·7H2O, 0.2 g / L of Biotin, and 5 mL / L of H2SO4.

7. Xylanase prepared by the method according to claim 6.

8. Application of the recombinant plasmid pSc048 according to any one of claims 1-3, the genetically engineered bacterium according to claim 4, the construction method according to claim 5, and the xylanase according to claim 7 in degrading straw-like agricultural waste.

9. The application according to claim 8, characterized in that, The straw-like agricultural waste is corn cob, sugarcane bagasse, cottonseed hull or rape straw.

10. The application according to claim 8, wherein The application comprises the following specific steps: adding lignocellulose into a sodium acetate buffer solution with a pH of 5.0, and then adding the xylanase according to claim 7, and reacting at 50°C.

11. Application of the sugar solution produced by the application according to any one of claims 8-10 in promoting plant growth. Preferably, the plant is cucumber or tomato.

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